Influence of maternal stress on uranium-induced developmental toxicity in rats

It has been demonstrated that uranium is an embryo/fetal toxicant when given orally or subcutaneously to pregnant mice. On the other hand, maternal stress has been shown to enhance the developmental toxicity of a number of metals. In this study, maternal toxicity and developmental effects of a concurrent exposure to uranyl acetate dihydrate (UAD) and restraint stress were evaluated in rats. Four groups of pregnant animals were given subcutaneous injections of UAD at 0.415 and 0.830 mg/kg/day on Days 6 to 15 of gestation. Animals in two of these groups were also subjected to restraint for 2 hr/day during the same gestational days. Control groups included restrained and unrestrained pregnant rats not exposed to UAD. Cesarean sections were performed on gestation Day 20, and the fetuses were weighed and examined for malformations and variations. Maternal toxicity and embryotoxicity were noted at 0.830 mg/kg/day of UAD, while fetotoxicity was evidenced at 0.415 and 0.830 mg/kg/day of UAD by significant reductions in fetal body weight and increases in the total number of skeletally affected fetuses. No teratogenic effects were noted in any group. Maternal restraint enhanced uranium-induced embryo/fetal toxicity only at 0.830 mg/kg/day, a dose that was also significantly toxic to the dams. As in previous studies with other metals, maternal stress enhances uranium-induced developmental toxicity at uranium doses that are highly toxic to the dams; however, at doses that are less acutely toxic the role of maternal stress would not be significant.     

Native-like cyclic peptide models of a viral antigenic site: finding a balance between rigidity and flexibility

Antigenic site A of foot-and-mouth disease virus (serotype C) has been reproduced by means of cyclic versions of peptide A15, YTASARGDLAHLTTT, corresponding to residues 136-150 of envelope protein VP1. A structural basis for the design of the cyclic peptides is provided by crystallographic data from complexes between the Fab fragments of anti-site A monoclonal antibodies and A15, in which the bound peptide is folded into a quasi-cyclic pattern. Head-to-tail cyclizations of A15 do not provide peptides of superior antigenicity. Internal disulfide cyclization, however, leads to analogs which are recognized as one to two orders of magnitude better than linear A15 in both ELISA and biosensor experiments. CD and NMR studies show that the best antigen, CTASARGDLAHLTT-Ahx-C (disulfide), is very insensitive to environment-induced conformational change, suggesting that cyclization helps to stabilize a bioactive-like structure.     

Use of the MIDI-FAME technique to characterize groundwater communities

Fatty acid methyl ester (FAME) profiles were identified directly from groundwater microbial communities concentrated on and extracted with polycarbonate filters. The sensitivity of this direct extraction method was determined using pure cultures of Acinetobacter junii, Pseudomonas putida and Stenotrophomonas maltophilia. A minimum concentration of 107 cells filter-1 was required to identify the predominant fatty acids from each culture. However, at least 3.7 x 109 cells filter-1 were required to obtain fatty acid profiles that matched the signature profiles for pure cultures in a commercial database. While several saturated fatty acids (i.e. 14 : 0, 16 : 0, 18 : 0) were extracted from the polycarbonate filters, they were readily subtracted from microbial fatty acid profiles and did not interfere with the characterization of pure cultures or environmental samples. For the environmental samples, 3 l of groundwater from the Savannah River Site, Aiken, SC, (USA) contained sufficient biomass for direct extraction. A comparative analysis of FAME groundwater profiles demonstrated a qualitative difference among communities sampled from spatially discrete locations, while a groundwater well that was sampled at two time points showed strong similarities over time. Concentration of microbial biomass on polycarbonate filters coupled with the MIDI-FAME extraction of both biomass and filter was a useful technique to characterize microbial communities from groundwater.     

Protein-protein interaction revealed by NMR T(2) relaxation experiments: the lipoyl domain and E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

T(2) relaxation experiments in combination with chemical shift and site-directed mutagenesis data were used to identify sites involved in weak but specific protein-protein interactions in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The pyruvate decarboxylase component, a heterotetramer E1(alpha(2)beta(2)), is responsible for the first committed and irreversible catalytic step. The accompanying reductive acetylation of the lipoyl group attached to the dihydrolipoyl acetyltransferase (E2) component involves weak, transient but specific interactions between E1 and the lipoyl domain of the E2 polypeptide chain. The interactions between the free lipoyl domain (9 kDa) and free E1alpha (41 kDa), E1beta (35 kDa) and intact E1alpha(2)beta(2) (152 kDa) components, all the products of genes or sub-genes over-expressed in Escherichia coli, were investigated using heteronuclear 2D NMR spectroscopy. The experiments were conducted with uniformly (15)N-labeled lipoyl domain and unlabeled E1 components. Major contact points on the lipoyl domain were identified from changes in the backbone (15)N spin-spin relaxation time in the presence and absence of E1(alpha(2)beta(2)) or its individual E1alpha or E1beta components. Although the E1alpha subunit houses the sequence motif associated with the essential cofactor, thiamin diphosphate, recognition of the lipoyl domain was distributed over sites in both E1alpha and E1beta. A single point mutation (N40A) on the lipoyl domain significantly reduces its ability to be reductively acetylated by the cognate E1. None the less, the N40A mutant domain appears to interact with E1 similarly to the wild-type domain. This suggests that the lipoyl group of the N40A lipoyl domain is not being presented to E1 in the correct orientation, owing perhaps to slight perturbations in the lipoyl domain structure, especially in the lipoyl-lysine beta-turn region, as indicated by chemical shift data. Interaction with E1 and subsequent reductive acetylation are not necessarily coupled.     

Memory in viral quasispecies

Biological adaptive systems share some common features: variation among their constituent elements and continuity of core information. Some of them, such as the immune system, are endowed with memory of past events. In this study we provide direct evidence that evolving viral quasispecies possess a molecular memory in the form of minority components that populate their mutant spectra. The experiments have involved foot-and-mouth disease virus populations with known evolutionary histories. The composition and behavior of the viral population in response to a selective constraint were influenced by past evolutionary history in a way that could not be predicted from examination of consensus nucleotide sequences of the viral populations. The molecular memory of the viral quasispecies influenced both the nature and the intensity of the response of the virus to a selective constraint.     

Comparative efficacy of several potential treatments for copper mobilization in copper-overloaded rats

d-Penicillamine (DPA) is effective in the treatment of Wilson’s disease, whereas zinc salts are also used as a therapy for this disorder of copper transport. Recently, it has been shown that the copper chelators 1,4,7,11-tetraazaundecane tetrahydrochloride (TAUD) and tetraethylenepentamine pentahydrochloride (TETREN) could be useful for copper mobilization in rats. Because these agents could be potential clinical alternatives to DPA for patients with Wilson’s disease who are intolerant to this drug, we examined whether oral administration of TAUD and TETREN could be effective in mobilizing copper in experimental copper-overloaded rats. The efficacy of a combined administration of zinc and DPA, TAUD, or TETREN was also assessed. Rats were copper loaded with 0.125% copper acetate in water for 12 wk. After this period, DPA, TAUD, and TETREN were administered by gavage at 0.67 mmol/kg/d for 5 d, and zinc was given at 2.5 mg Zn/kg/d. Twelve weeks of copper loading resulted in a 32-fold increase in total hepatic copper. TETREN was the most effective chelator in increasing the urinary excretion of copper. However, it did not reduce significantly the hepatic copper levels. In turn, combined administration of zinc and chelating agents significantly reduced the amount of copper found in the feces. Although TAUD and TETREN showed a similar or higher efficacy to DPA in mobilizing copper, concurrent treatment of chelating agents and zinc salts should be discarded according to the current results.     

Laying date and clutch size of Great Tits(Parus major) in the Mediterranean region: a comparison of four habitat types

Summary Laying data and clutch size of Great Tits were studied in four different habitats in eastern Spain: two holm oak(Quercus ilex) forests, at 500 and 900–950 m a.s.l., a zeen oak(Quercus faginea) forest, at 900–1100 m a.s.l., a pine(Pinus sylvestris) forest, at 1000–1050 m a.s.l., and orange(Citrus aurantium) plantations, at 30 m a.s.l. All sites were placed at about the same latitude (39–41°N), and all were studied during the same years (1992–95). Our results show that (1) laying date did not differ between the natural habitats at the same altitude (range of the means of yearly means 4–8 May); (2) within the same habitat type (holm oak forest) laying date was earlier at low altitude (30 Aprilvs. 8 May); (3) laying date was earlier in the orange plantations (21 April) than in natural habitats; (4) among natural habitats at the same altitude, clutch size decreased from zeen oak (mean of yearly means 7.3 eggs) to holm oak (7.0 eggs) to pine forests (6.4 eggs), though only the difference between zeen oak and pine forests was significant; (5) within the same habitat type (holm oak forest), the clutch size tended to be larger at high altitude (7.0vs. 5.9 eggs); and (6) clutch size in orange plantations (7.7 eggs) did not differ significantly from that of the zeen oak forest, but was larger than in the holm oak and pine forests. We discuss the effect of the habitat type on laying date and clutch size of Great Tits.

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Legedatum und Gelegegröße der Kohlmeise(Parus major) in mediterranen Gebieten: Ein Vergleich zwischen vier verschiedenen Biotopen

Zusammenfassung Legedatum und Gelegegröße der Kohlmeise wurden in vier unterschiedlichen Biotopen in Ostspanien untersucht: zwei Steineichenwälder(Quercus ilex) in 500 m und 900–950 mNN, ein Bergeichenwald(Quercus faginea) in 900–1100 mNN, ein Kiefernwald(Pinus sylvestris) in 1000–1050 mNN und eine Orangenpflanzung(Citrus aurantium) in 30 mNN. Alle fünf Gebiete lagen auf etwa demselben Breitengrad (39–41°N) und wurden 1992–1995 parallel untersucht.(1) Auf gleicher Meereshöhe unterscheidet sich der Legebeginn nicht zwischen den verschiedenen Waldbiotopen (im Mittel 4.–8. Mai). (2) Innerhalb desselben Biotoptyps (Steineichenwald) war der Legebeginn auf niedrigerer Meereshöhe früher als in höheren Lagen (30. April vs. 8. Mai). (3) Im Orangenhain wurde früher mit der Eiablage begonnen (21. April) als in den Waldbiotopen. (4) Auf gleicher Meereshöhe nahm die Gelegegröße vom Bergeichenwald (Mittelwert 7.3 Eier) über die Steineichenwälder (7,0 Eier) zum Kiefernwald hin ab (6.4 Eier), jedoch ist nur der Unterschied zwischen Bergeichenwald und Kiefernwald signifikant. (5) Innerhalb der Steineichenwälder besteht die Tendenz zu größerer Gelegegröße in den höheren Lagen (7.0 vs. 5.9 Eier). (6) Im Orangenhain war die Gelegegröße mit durchschnittlich 7.7 Eier ähnlich der im Bergeichenwald, aber größer als in den Steineichen- und Kiefernwälder.

     

Regulation of Multidrug Resistance Proteins by Genistein in a Hepatocarcinoma Cell Line: Impact on Sorafenib Cytotoxicity

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 μM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 μM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 μM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 μM) and MRP2 induction by GNT (only at 10 μM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements.     

Artery Wall Assessment Helps Predict Kidney Transplant Outcome

Background

Kidney transplant recipients have high cardiovascular risk, and vascular inflammation may play an important role. We explored whether the inflammatory state in the vessel wall was related to carotid intima-media thickness (c-IMT) and patient survival following kidney transplantation.

Methods

In this prospective observational cohort study we measured c-IMT and expression of proinflammatory cytokines and adhesion molecules in the inferior epigastric artery in 115 kidney transplant candidates. Another c-IMT measurement was done 1-year post-transplantation in 107. By stepwise multiple regression analysis we explored factors associated with baseline c-IMT and their changes over time. Multivariate Cox regression analysis was constructed to identify risk factors for mortality.

Results

A worse cardiovascular profile (older age, smoker, diabetic, carotid plaque, systolic blood pressure and vascular calcification) and higher VCAM-1 levels were found in patients in the highest baseline c-IMT tertile, who also had a worse survival. Factors independently related to baseline c-IMT were age (β=0.369, P<0.0001), fasting glucose (β=0.168, P=0.045), smoking (β=0.228, P=0.003) and VCAM-1 levels (β=0.244, P=0.002). Independent factors associated with c-IMT measurement 1-year post-transplantation were baseline c-IMT (β=-0.677, P<0.0001), post-transplant diabetes (β=0.225, P=0.003) and triglycerides (β=0.302, P=0.023). Vascular VCAM-1 levels were associated with increased risk of mortality in bivariate and multivariate Cox regression. Notably, nearly 50% of patients showed an increase or maintenance of high c-IMT 1 year post-transplantation and these patients experienced a higher mortality (13 versus 3.5%; P=0.021).

Conclusion

A worse cardiovascular profile and a higher vascular VCAM-1 protein levels at time of KT are related to subclinical atheromatosis. This could lead to a higher post-transplant mortality. Pre-transplant c IMT, post-transplant diabetes and triglycerides at 1-year post-transplantation may condition a high c-IMT measurement post-transplantation, which may decrease patient survival.