In situ hybridization to somatic metaphase chromosomes of potato

Summary An in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a ladderpattern in genomic Southern's of Solanum tuberosum and Solanum phureja, but not in those of Solanum brevidens and two Nicotiana species. This sequence hybridized predominantly on telomeric and centromeric regions of all chromosomes, although chromosomes 7, 8, 10 and 11 were not always labeled clearly.     

Fusion of small unilamellar vesicles with viable EDTA-treated Escherichia coli cells.

Fusion characteristics of EDTA-treated Escherichia coli cells with small unilamellar vesicles were investigated, using a membrane fusion assay based on resonance energy transfer. Ca2+-EDTA treatments of Escherichia coli O111:B4 (wild type), E. coli C600 (rough), and E. coli D21f2 (deep rough) which permeabilize the outer membrane by inducing the release of lipopolysaccharide and outer membrane proteins resulted in fusion activity of the intact and viable bacteria with small unilamellar vesicles. No fusion activity was observed when the EDTA treatment was omitted. Fusion could be elicited at low pH and by a combination of a higher pH and Ca2+. The low-pH-induced fusion was composed of a fast and a slow reaction. The latter and the Ca2+-induced fusion could be completely inhibited by trypsin treatments of the EDTA-treated cells, which also resulted in the simultaneous disappearance of two outer membrane protein bands (50 and 58 kilodaltons) and the appearance of proteins banding at 22, 52, and 54 kilodaltons. The most efficient fusion was obtained with negatively charged liposomes composed of cardiolipin. In contrast to the Ca2+-induced fusion, fusion was observed at low pH with small unilamellar vesicles containing lipids with decreased negative charge (phosphatidylserine). Fluorescent and phase-contrast microscopy revealed that essentially all bacteria were engaged in fusion. We propose that a Ca2+-EDTA treatment of E. coli cells results in the appearance of phospholipids and the exposure of a protein(s) in the outer leaflet of the outer membrane, both of which could mediate fusion with liposomes.     

Projected climate change causes loss and redistribution of genetic diversity in a model metapopulation of a medium‐good disperser

Climate change causes species ranges to shift geographically as individuals colonise new suitable temperature zones or fail to reproduce where climate conditions fall below tolerance levels. Little is known about the potential loss of genetic diversity in such dynamic ranges. We investigated the level and distribution of neutral genetic diversity in shifting metapopulations during three scenarios of temperature increase projected for this century and at various degrees of weather variability. We used an individual‐based and spatially explicit metapopulation model in which temperature zones were simulated to move across a fragmented landscape following different climate change scenarios. Although the connectivity between habitat patches allowed the species, modelled after the middle spotted woodpecker Dendrocopos medius, to move along with the shifting temperature range, existing neutral genetic diversity was lost under all three temperature increase scenarios. This was independent of the loss of individuals. The explanation for this effect is that only a part of the original genetic variation moved into the newly colonised habitat. Under increased weather variability the number of individuals and the number of alleles per locus were persistently lower. However, the pattern of changes in allele distributions under temperature zone shifts was the same under all weather variability levels. Genetic differentiation between populations had a tendency to increase at metapopulation range margins, but decreased again when population sizes increased in time. Increased weather variability led to increased variation around the mean genetic differentiation across the metapopulation. Our results illustrate the usefulness of more realistic models for studying the effects of climate change on metapopulations. They indicate that biodiversity monitoring indices based on species occurrence and abundance are not a good proxy for the trend in the level of genetic diversity. Further, the results underline the importance of conserving areas where species have existed for a long time as modern refugia for genetic diversity.     

Ueber Virulenz und Virulenz-Curven

Zusammenfassung Es wird eine Methode angegeben, um die Virulenz graphisch darzustellen. Der Verlauf der Curve wird durch Aenderung der Factoren sowohl des Erregers als auch des Wirtes deutlich beeinflusst.     

Conserving biodiversity efficiently: what to do, where, and when

Conservation priority-setting schemes have not yet combined geographic priorities with a framework that can guide the allocation of funds among alternate conservation actions that address specific threats. We develop such a framework, and apply it to 17 of the world's 39 Mediterranean ecoregions. This framework offers an improvement over approaches that only focus on land purchase or species richness and do not account for threats. We discover that one could protect many more plant and vertebrate species by investing in a sequence of conservation actions targeted towards specific threats, such as invasive species control, land acquisition, and off-reserve management, than by relying solely on acquiring land for protected areas. Applying this new framework will ensure investment in actions that provide the most cost-effective outcomes for biodiversity conservation. This will help to minimise the misallocation of scarce conservation resources.     

Alterations in aortic antioxidant components in an experimental model of atherosclerosis: A time-course study

Antioxidant component alterations in the aorta during atherogenesis were examined in atherosclerosis-susceptible (SUS) Japanese quail fed a cholesterol-supplemented (0.5% w/w) diet. Birds fed a non-supplemented diet provided information on the effects of aging on endogenous antioxidants. One hundred adult SUS males were used. Birds were sacrificed after 0, 4, 8 and 12 weeks on the diets and were examined for plaque development and corresponding antioxidant component alterations in aorta and myocardium. With aging, superoxide dismutase (SOD) activity was increased in both tissues, whereas aortic glutathione peroxidase (GPx) activity and myocardial glutathione reductase (GRd) activity decreased. Myocardial ascorbate levels increased with aging, with a reciprocal decrease in myocardial tocopherol levels. Following 4 weeks of cholesterol supplementation, aortic GRd decreased, SOD activity increased, but activities of GPx and catalase were unchanged. This same qualitative pattern of antioxidant enzyme changes was also found in myocardium. Thus, although aortic antioxidant enzyme changes produced by cholesterol feeding and aging showed some similarities, the early phase of atherogenesis does not simply reflect accelerated aging. In the late stages of atherogenesis, SOD activity returned to baseline, but other antioxidant enzymes remained unaltered from levels characterizing the early phase of lesion development. There was no detectable functional coupling between changes in GPx and GRd, nor between SOD (which produces hydrogen peroxide) and GPx or catalase (which utilize hydrogen peroxide as substrate). Previously reported alterations in erythrocyte antioxidant enzyme components during atherogenesis in quail were not predictive of changes in the corresponding enzymes in the aorta and myocardium.     

Effect of dietary selenium concentration and duration of selenium feeding on hepatic glutathione concentrations in rats

Studies were conducted in rats to determine the effect of dietary selenium (Se) concentration on hepatic glutathione concentrations and enzyme activities associated with the maintenance of the cellular glutathione status. Male rats were fed 0.1, 3.0, or 6.0 ppm Se as Na2SeO3 for 2, 4, or 6 weeks at which time they were killed and analyses were performed. Both 3.0 and 6.0 ppm Se caused a significant dose-dependent increase in hepatic-reduced glutathione (GSH) by 4 weeks of feeding compared to 0.1 ppm Se. The increase in GSH was preceded by significant, dose-dependent increases in oxidized glutathione (GSSG) as well as the GSSG to GSH ratio. Increases in GSSG and the GSSG to GSH ratio as well as in glutathione reductase and glucose-6-phosphate dehydrogenase activities were observed by 2 weeks of high Se feeding. The current findings substantiate previous results demonstrating effects of high Se on hepatic glutathione concentrations (R. A. LeBoeuf and W. G. Hoekstra, J. Nutr. 113:845-854, 1983) and further suggest that increased cellular GSSG concentrations or the GSSG to GSH ratio caused by 3.0 and 6.0 ppm dietary Se signals for "adaptive" changes in hepatic glutathione metabolism.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Can Nep1-like proteins form oligomers?

Nep1-like proteins (NLPs) are a novel family of microbial elicitors of plant necrosis that induce a hypersensitive-like response in dicot plants. The spatial structure and role of these proteins are yet unknown. In a paper published in BMC Plant Biology (2008; 8:50) we have proposed that the core region of Nep1-like proteins (NLPs) belong to the Cupin superfamily. Based on what is known about the Cupin superfamily, in this addendum to the paper we discuss how NLPs could form oligomers.Key words: quaternary structure, necrosis and ethylene inducing proteins, NLPs, MpNEP1, MpNEP2, NPP1, Moniliophthora perniciosa, Phytophthora parasiticaCupins may be organized as monomers, dimers, hexamers and octamers of β-barrel domains.¹ To the best of our knowledge trimers have not been detected yet. The interaction of two monomers building up a dimeric structure is basically performed by three types of interactions: hydrophobic interactions between β-strands in different subunits, salt bridges and hydrogen bonds between β-strands. In cupin dimers, the hydrophobic interactions occur between two βI strands in different subunits (Fig. 1A and B). This strand represents the central axis of rotation of the dimer as one residue in βI interacts with the corresponding residue in the other subunit (Fig. 1B). Therefore, all residues in βI must be hydrophobic, as one residue interacts with the other subunit and the next one in the sequence interacts with the interior of the protein. Charged residues in βI would disrupt such interactions. Most cupin dimers have strong hydrophobic residues such as tryptophan (W), phenylalanine (F) and methionine (M) pointing towards the own subunit (↓), while small hydrophobic residues such as leucine (L), isoleucine (I), and valine (V) point to the other subunit (↑). A particular case is leucine that interacts with other subunits, for instance, βI = liaW (positions 217–220 in Fig. 1B) and βI = LVsw of type I and II NLP consensuses, respectively. Therefore, the pattern of hydropathicity suggests that the side chain orientation is βI = l217 ↑ i218 ↓ a219 ↑ W220 ↓ d221 ↑. However we observe that just after βI there is a charged residue (aspartate D221) which would point outwards disrupting the dimer or at least making it less stable. It is interesting to observe that the requirement for a negatively charged residue at this last position is very high: 96% of all type I NLPs contains an aspartate (D) or glutamate (E) indicating an important role for it, maybe in avoiding dimerization of the NLPs. A second interesting hypothesis is as follows: several cupins are oxygenases, decarboxylases, etc. and use a negatively charged residue, such as aspartate or glutamate as proton donor.¹ Now, if the alternate pattern of side chains of the residues is βI = l217 ↓ i218 ↑ a219 ↓ W220 ↑ d221 ↓, instead of the previous one, then the aspartate or glutamate residue would point to the hydrophobic pocket and would be positioned to interact with the metal ion, as in cupins with enzymatic activity. However, there are no experimental evidences that the NLPs have enzymatic activity.Open in a separate windowFigure 1(A) Three-dimensional structure prediction for type I NLP consensus, (B) Interface between two βI strands in type I NLP consensus. From the left to the right: EF-coil with the conserved residue H162, βC and βH strands (superposed) with the conserved histidines H133 and H135 in βC, H193 and leucine L195 in βH, W220 in βI and W118 in βB. The strands in the right subunit follow the same pattern but rotated.The second type of interaction is salt bridges between charged residues in different subunits. Analyzing all interacting side chains in the 1VJ2 protein (dimer), we verify that the charged side chains of N35 and E57 (numbers in original structure) are only 2.72 Å apart. In the NLPs, this corresponds to N108^36% (Q108^60%) at the border of βB and E138^98%. The negatively charged residue D125 helps to correct the orientation of the subunits in relation to each other avoiding any disorientation. The high conservation level of these residues suggests that NLPs are dimeric structures. However, as we will see next, only hydrophobic and charged interactions are not enough to build a dimer.Garcia et al. (2007)² have used small angle X-ray scattering (SAXS) to show that, in solution, at low concentrations (<2 mg/ml) the two copies of the NLPs of Moniliophthora perniciosa, MpNEP1 and MpNEP2, exist as dimers and monomers, respectively. The same technique showed that at higher concentrations, >5 mg/ml, both proteins exist as dimers, as is the case for PpNPP1.² They also reported, based on electrophoresis analysis, that PpNPP1 and MpNEP1 exist as oligomers and MpNEP2 as monomers.² However, experiments with the PpNPP1 in size exclusion chromatography using myoglobin as size standard suggest that PpNPP1 is a monomer.³ Figure 2 compares MpNEP1, MpNEP2 and PpNPP1, where the most relevant differences in sequence are marked with asterisks (*) and are possibly related to the differences in oligomeric properties between MpNEP1 and PpNPP1 with MpNEP2. These positions are methionine M27 and leucine L35, which occur only in MpNEP2, glycine G250, which occurs only in MpNEP2 and NEP1 (Fusarium oxysporum) and lysine K31, which occurs only MpNEP2, {"type":"entrez-protein","attrs":{"text":"BAB04114","term_id":"10173008"}}BAB04114 (Bacillus halodurans) and {"type":"entrez-protein","attrs":{"text":"AAU23136","term_id":"52003194"}}AAU23136 (Bacillus licheniformis). The other residues are aspartate D28, which occurs 9 times and alanine A37 which occurs 7 times of all investigated NLPs. Thus, the sequence mdHDkiakl at the start of the NLPs seems to explain the monomeric state of MpNEP2, although at higher concentrations they form dimers. Besides the weak hydrophobic interactions, dimeric cupins and bicupins (two β barrels in the same sequence building up a dimeric-like 4d-structure) are stable structures (see Fig. 1 in ref. ⁴). By aggregating the first β-strand in the start domain of one β-barrel to the ABIDG β-sheet of the other β-barrel, composing a big ABIDGY β-sheet (Y is the first β-strand). For instance, using the bicupin 1L3J (oxalate decarboxylase) as template, the low confidence level β-strand at position 26–33 (v in H29D30 avv) in type I NLPs corresponds to the first β-strand. Since this proceeds from both barrels they can build a stable structure (see Fig. 1 in ref. ⁴). The quaternary structure is related to the presence of interaction residues in the BID β-sheet of the cupin structure. These are present in the NLPs and would enable them to form dimers.Open in a separate windowFigure 2Alignment of type I NLP consensus, PpNPP1, MpNEP1 and MpNEP2. Solid line boxes are β-strands, double line boxes are α-helices. The sequence positions marked with asterisks (*) are possibly related to the differences in oligomeric properties between MpNEP1 and PpNPP1 with MpNEP2.     

Scavenger receptor class B type I-mediated uptake of serum cholesterol is essential for optimal adrenal glucocorticoid production

Impaired scavenger receptor class B type I (SR-BI)-mediated uptake of HDL-cholesterol esters (HDL-CE) induces adrenal insufficiency in mice. Humans contain an alternative route of HDL-CE clearance, namely through the transfer by cholesteryl ester transfer protein (CETP) to apolipoprotein B lipoproteins for subsequent uptake via the LDL receptor. In this study, we determined whether CETP can compensate for loss of adrenal SR-BI. Transgenic expression of human CETP (CETP Tg) in SR-BI knockout (KO) mice increased adrenal HDL-CE clearance from 33–58% of the control value. SR-BI KO/CETP Tg and SR-BI KO mice displayed adrenal hypertrophy due to equally high plasma adrenocorticotropic hormone levels. Adrenal cholesterol levels and plasma corticosterone levels were 38–52% decreased in SR-BI KO mice with and without CETP expression. SR-BI KO/CETP Tg mice also failed to increase their corticosterone level after lipopolysaccharide challenge, leading to an identical >4-fold increased tumor necrosis factor-α response compared with controls. These data indicate that uptake of CE via other routes than SR-BI is not sufficient to generate the cholesterol pool needed for optimal adrenal steroidogenesis. In conclusion, we have shown that CETP-mediated transfer of HDL-CE is not able to reverse adrenal insufficiency in SR-BI knockout mice. Thus, SR-BI-mediated uptake of serum cholesterol is essential for optimal adrenal function.