The role of apolipoprotein E in the elimination of liposomes from blood by hepatocytes in the mouse

We evaluated the role of apolipoprotein E (apoE) in the clearance of neutral and negatively charged liposomes by hepatocytes in apoE-deficient mice. Negatively charged liposomes were cleared at identical rates in apoE-deficient and wild-type mice; neutral liposomes were cleared at a 3.6-fold slower rate in apoE-deficient mice. ApoE deficiency did not affect hepatic uptake of negatively charged liposomes but lowered that of neutral liposomes >5-fold. Hepatocyte uptake of neutral liposomes was reduced >20-fold in apoE-deficient mice; that of negatively charged liposomes remained unchanged. We conclude that uptake of neutral liposomes by hepatocytes is nearly exclusively apoE-mediated.     

Significance tests and weighted values for AFLP similarities, based on Arabidopsis in silico AFLP fragment length distributions

Many AFLP studies include relatively unrelated genotypes that contribute noise to data sets instead of signal. We developed: (1) estimates of expected AFLP similarities between unrelated genotypes, (2) significance tests for AFLP similarities, enabling the detection of unrelated genotypes, and (3) weighted similarity coefficients, including band position information. Detection of unrelated genotypes and use of weighted similarity coefficients will make the analysis of AFLP data sets more informative and more reliable. Test statistics and weighted coefficients were developed for total numbers of shared bands and for Dice, Jaccard, Nei and Li, and simple matching (dis)similarity coefficients. Theoretical and in silico AFLP fragment length distributions (FLDs) were examined as a basis for the tests. The in silico AFLP FLD based on the Arabidopsis thaliana genome sequence was the most appropriate for angiosperms. The G + C content of the selective nucleotides in the in silico AFLP procedure significantly influenced the FLD. Therefore, separate test statistics were calculated for AFLP procedures with high, average, and low G + C contents in the selective nucleotides. The test statistics are generally applicable for angiosperms with a G + C content of approximately 35-40%, but represent conservative estimates for genotypes with higher G + C contents. For the latter, test statistics based on a rice genome sequence are more appropriate.     

Physiological and gene expression analysis of inhibition of Desulfovibrio vulgaris hildenborough by nitrite

A Desulfovibrio vulgaris Hildenborough mutant lacking the nrfA gene for the catalytic subunit of periplasmic cytochrome c nitrite reductase (NrfHA) was constructed. In mid-log phase, growth of the wild type in medium containing lactate and sulfate was inhibited by 10 mM nitrite, whereas 0.6 mM nitrite inhibited the nrfA mutant. Lower concentrations (0.04 mM) inhibited the growth of both mutant and wild-type cells on plates. Macroarray hybridization indicated that nitrite upregulates the nrfHA genes and downregulates genes for sulfate reduction enzymes catalyzing steps preceding the reduction of sulfite to sulfide by dissimilatory sulfite reductase (DsrAB), for two membrane-bound electron transport complexes (qmoABC and dsrMKJOP) and for ATP synthase (atp). DsrAB is known to bind and slowly reduce nitrite. The data support a model in which nitrite inhibits DsrAB (apparent dissociation constant K(m) for nitrite = 0.03 mM), and in which NrfHA (K(m) for nitrite = 1.4 mM) limits nitrite entry by reducing it to ammonia when nitrite concentrations are at millimolar levels. The gene expression data and consideration of relative gene locations suggest that QmoABC and DsrMKJOP donate electrons to adenosine phosphosulfate reductase and DsrAB, respectively. Downregulation of atp genes, as well as the recorded cell death following addition of inhibitory nitrite concentrations, suggests that the proton gradient collapses when electrons are diverted from cytoplasmic sulfate to periplasmic nitrite reduction.     

Folate deprivation results in the loss of breast cancer resistance protein (BCRP/ABCG2) expression. A role for BCRP in cellular folate homeostasis

Breast cancer resistance protein (BCRP/ABCG2) is currently the only ABC transporter that exports mono- and polyglutamates of folates and methotrexate (MTX). Here we explored the relationship between cellular folate status and BCRP expression. Toward this end, MCF-7 breast cancer cells, with low BCRP and moderate multidrug resistance protein 1 (MRP1/ABCC1) levels, and their mitoxantrone (MR)-resistant MCF-7/MR subline, with BCRP overexpression and low MRP1 levels, were gradually deprived of folic acid from 2.3 microm to 3 nm resulting in the sublines MCF-7/LF and MCF-7/MR-LF. These cell lines expressed only residual BCRP mRNA and protein levels and retained a poor MRP2 (ABCC2) through MRP5 (ABCC5) expression. Furthermore, MCF-7/MR-LF cells also displayed 5-fold decreased MRP1 levels relative to MCF-7/MR cells. In contrast, BCRP overexpression was largely retained in MCF-7/MR cells grown in MR-free medium containing 2.3 microm folic acid. Loss of BCRP expression in MCF-7/LF and MCF-7/MR-LF cells resulted in the following: (a) a prominent decrease in the efflux of Hoechst 33342, a BCRP substrate; (b) an approximately 2-fold increase in MR accumulation as revealed by flow cytometry; this was accompanied by a 2.5- and approximately 84-fold increased MR sensitivity in these cell lines, respectively. Consistently, Ko143, a specific BCRP inhibitor, rendered MCF-7 and MCF-7/MR cells 2.1- and approximately 16.4-fold more sensitive to MR, respectively. Loss of BCRP expression also resulted in the following: (c) an identical MTX sensitivity in these cell lines thereby losing the approximately 28-fold MTX resistance of the MCF-7/MR cells; (d) an approximately 2-fold increase in the 4- and 24-h accumulation of [(3)H]folic acid. Furthermore, MCF-7/MR-LF cells displayed a significant increase in folylpoly-gamma-glutamate synthetase activity. Hence, consistent with the mono- and polyglutamate folate exporter function of BCRP, down-regulation of BCRP and increased folylpoly-gamma-glutamate synthetase activity appear to be crucial components of cellular adaptation to folate deficiency conditions. This is the first evidence for the possible role of BCRP in the maintenance of cellular folate homeostasis.     

The X-ray structure of trans-3-chloroacrylic acid dehalogenase reveals a novel hydration mechanism in the tautomerase superfamily

Isomer-specific 3-chloroacrylic acid dehalogenases function in the bacterial degradation of 1,3-dichloropropene, a compound used in agriculture to kill plant-parasitic nematodes. The crystal structure of the heterohexameric trans-3-chloroacrylic acid dehalogenase (CaaD) from Pseudomonas pavonaceae 170 inactivated by 3-bromopropiolate shows that Glu-52 in the alpha-subunit is positioned to function as the water-activating base for the addition of a hydroxyl group to C-3 of 3-chloroacrylate and 3-bromopropiolate, whereas the nearby Pro-1 in the beta-subunit is positioned to provide a proton to C-2. Two arginine residues, alphaArg-8 and alphaArg-11, interact with the C-1 carboxylate groups, thereby polarizing the alpha,beta-unsaturated acids. The reaction with 3-chloroacrylate results in the production of an unstable halohydrin, 3-chloro-3-hydroxypropanoate, which decomposes into the products malonate semialdehyde and HCl. In the inactivation mechanism, however, malonyl bromide is produced, which irreversibly alkylates the betaPro-1. CaaD is related to 4-oxalocrotonate tautomerase, with which it shares an N-terminal proline. However, in 4-oxalocrotonate tautomerase, Pro-1 functions as a base participating in proton transfer within a hydrophobic active site, whereas in CaaD, the acidic proline is stabilized in a hydrophilic active site. The altered active site environment of CaaD thus facilitates a previously unknown reaction in the tautomerase superfamily, the hydration of the alpha,beta-unsaturated bonds of trans-3-chloroacrylate and 3-bromopropiolate. The mechanism for these hydration reactions represents a novel catalytic strategy that results in carbon-halogen bond cleavage.     

Facilitated drug influx by an energy-uncoupled secondary multidrug transporter

The majority of bacterial multidrug resistance transporters belong to the class of secondary transporters. LmrP is a proton/drug antiporter of Lactococcus lactis that extrudes positively charged lipophilic substrates from the inner leaflet of the membrane to the external medium. This study shows that LmrP is a true secondary transporter. In the absence of a proton motive force, LmrP facilitates downhill fluxes of ethidium in both directions. These fluxes are inhibited by other substrates of LmrP. The cysteine-reactive agent p-chloromercuri-benzene sulfonate inhibits these fluxes in wild type LmrP but not in the cysteine-less LmrP C270A mutant. Cysteine mutagenesis of LmrP resulted in three mutants, D68C/C270A, D128C/C270A, and E327C/C270A, with an energy-uncoupled phenotype. Asp68 is located in the conserved motif GXXX(D/E)(R/K)XGRK for the major facilitator superfamily of secondary transporters and was found to play an important role in energy coupling, whereas the negatively charged residues Asp128 and Glu327 have indirect effects on the transport process. L. lactis strains expressing these uncoupled mutants of LmrP show an increased rate of ethidium influx and an increased drug susceptibility compared with cells harboring an empty vector. The rate of influx in these mutants is enhanced by a transmembrane electrical potential, inside negative. These observations suggest a new strategy for eliminating drug-resistant microbial pathogens, i.e. the design and use of modulators of secondary multidrug resistance transporters that uncouple drug efflux from proton influx, thereby allowing transmembrane electrical potential-driven influx of cationic drugs.     

An in vitro system for the long-term tissue culture of juvenile rainbow trout (Oncorhynchus mykiss) testis

In vitro cultivation of fish gonad fragments continues to be an important experimental approach to answer both fundamental and applied scientific questions. The aim of the present investigation was to test whether juvenile rainbow trout (Oncorhynchus mykiss) testes cultured for a week or more were physiologically competent. Trout testis fragments (approximately 1 mm2) were placed on pieces of flat, culture plate insert filter, in a drop of liquid medium (modified Leibovitz's L-15), and floated on the medium surface in a multi-well culture plate. Culture plates were covered and incubated in air at 15 degrees C. Three different endpoints were used to test whether cultured testis fragments remained healthy and functional. First, a comparison of the histological appearance of testis fragments cultured in vitro for different periods up to 8 days with intact testes indicated that no differences were evident. Secondly, testis fragments incubated in medium supplemented with bovine calf serum (BCS) at concentrations of 2.5% and 25% BCS had significantly greater proliferation of interstitial and spermatocyst cells as measured by nuclear 5-bromo-2'-deoxyuridine incorporation. Finally, testis fragments cultured for 5 days and transplanted back into the donor fish underwent precocious sexual maturity in response to a regime of salmon pituitary extract injections and produced fertile sperm as determined by their ability to successfully fertilize eggs. These experiments demonstrate the utility of this method for in vitro culture of juvenile rainbow trout testis fragments.     

Comparative studies on the effect of ergot contaminated feed on performance and health of piglets and chickens

Two dose response trials were conducted with piglets and chickens to study the effects of increasing amounts of ergot (Claviceps purpurea) with a defined alkaloid content and pattern on performance, biochemical serum characteristics and organ weights (of chickens). The ergot was mixed into the cereal-soybean meal based diets at levels of 0, 0.5, 1, 2 and 4 g/kg. The total alkaloid content of the ergot was analysed to be 2775 mg/kg and showed the following composition: ergometrine 8.1%, ergotamine 5.4%, ergocomine 3.2%, alpha-ergocryptine 1.9%, ergocristine 14.9% and residue 66.5%. Each treatment was tested with eight castrated male and eight female piglets over a period of 35 days (8 kg initial live weight) and 28 male chickens for 21 days (43 g initial live weight). Cumulative daily dry matter intake and live weight gain [g/d] were 595, 535, 560, 577 and 490 and 413, 399, 420, 443 and 347 for the piglets fed the unsupplemented control diet and the diets containing 0.5, 1, 2 and 4 g ergot per kg, respectively. Feed intake and live weight gain of the piglets fed the highest ergot supplemented diet were significantly decreased. Serum aspartate aminotransferase activity of the 4 g ergot treatment was significantly increased. Also serum albumin concentrations showed significant linear alterations. Serum activities of glutamate dehydrogenase, gamma-glutamyltransferase, total protein and porcine growth hormone were not significantly influenced by dietary treatment. The experiment with chickens demonstrated no significant effects on performance due to dietary ergot exposure. The serum activities of glutamate dehydrogenase and alanine aminotransferase were not significantly influenced by dietary treatment while serum activities of gamma-glutamyltransferase and aspartate aminotransferase and the concentrations of albumin and total bilirubin were significantly affected. Heart weights showed a significant linear decrease due to ergot feeding. According to these results, piglets seemed to react more sensitively on the occurrence of ergot in the diet as compared to chickens. The critical level of total ergot alkaloids for piglets seemed to be in the range from 5.6 mg to 11.1 mg/kg diet for the present study. Ergot effects on signs of inflammation in the proximal duodenum occurred in chickens fed diets containing 2.8 mg and 11.1 mg total ergot alkaloids/kg although live performance remained unaffected. Further studies are necessary to define the critical level of ergot alkaloids in dependence on alkaloid pattern.     

Flow cytometry-assisted cloning of specific sequence motifs from complex 16S rRNA gene libraries

A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.