Tracing B-genome chromatin in Brassica napus x B. juncea interspecific progeny.

We used polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques to demonstrate the presence of Brassica B-genome chromosomes and putative B-genome introgressions in B. napus x B. juncea interspecific progeny. The B-genome--specific repeat sequence pBNBH35 was used to generate PCR products and FISH probes. The highest frequencies of viable progeny were obtained when B. napus was the maternal parent of the interspecific hybrid and the first backcross. B-genome--positive PCR assays were found in 34/51 fertile F2 progeny (67%), which was more than double the proportion found in fertile BC(1) progeny. Four B-genome--positive F(2)-derived families and 1 BC(1)-derived family were fixed or segregating for B. juncea morphology in the F(4) and BC(1)S(2), respectively, but in only 2 of these families did B. juncea-type plants exhibit B. juncea chromosome count (2n = 36) and typical B-genome FISH signals on 16 chromosomes. The remaining B. juncea-type plants had B. napus chromosome count (2n = 38) and no B-genome FISH signals, except for 1 exceptional F(4)-derived line that exhibited isolated and weak B-genome FISH signals on 11 chromosomes and typical A-genome FISH signals. B. juncea morphology was associated with B-genome--positive PCR signals but not necessarily with 16 intact B-genome chromosomes as detected by FISH. B-genome chromosomes tend to be eliminated during selfing or backcrossing after crossing B. juncea with B. napus, and selection of lines containing B-genome chromatin during early generations would be promoted by use of this B-genome repetitive marker.     

Phosphorus mining efficiency declines with decreasing soil P concentration and varies across crop species

High soil P concentrations hinder ecological restoration of biological communities typical for nutrient-poor soils. Phosphorus mining, i.e., growing crops with fertilization other than P, might reduce soil P concentrations. However, crop species have different P-uptake rates and can affect subsequent P removal in crop rotation, both of which may also vary with soil P concentration. In a pot experiment with three soil-P-levels (High-P: 125–155 mg P_Olsen/kg; Mid-P: 51–70 mg P_Olsen/kg; Low-P: 6–21 mg P_Olsen/kg), we measured how much P was removed by five crop species (buckwheat, maize, sunflower, flax, and triticale). Total P removal decreased with soil-P-level and depended upon crop identity. Buckwheat and maize removed most P from High-P and Mid-P soils and triticale removed less P than buckwheat, maize, and sunflower at every soil-P-level. The difference in P removal between crops was, however, almost absent in Low-P soils. Absolute and relative P removal with seeds depended upon crop species and, for maize and triticale, also upon soil-P-level. None of the previously grown crop species significantly affected P removal by the follow-up crop (perennial ryegrass). We can conclude that for maximizing P removal, buckwheat or maize could be grown.     

Single cell and tissue specific methods for evaluation of radiation and microgravity effects

A discussion of different methods to evaluate dose/response and biological effects of ionizing radiation is given. Confocal scanning laser microscopy (CSLM) is presented as a high performing observation method for evaluating different cytological effects. Standard cytochemical techniques can be used to analyse the cell in situ with minimal disturbance of morphology and structure. If a relatively small number of cells are affected by the treatment, the use of confocal microscope observations is fast and has a better resolution than conventional fluorescence microscopy. The optical sectioning capability of the CSLM makes it possible to analyse stacks of cells on detectors up to a depth of 200 micrometer with a resolution of 0.7 micrometer. This is used to analyse single cell electrophoresis results and nuclear track analysis in poly allyl diglycol carbonate (PADC). Consecutive analysis of cells cultivated on PADC, and analysis of nuclear tracks after chemical etched tracks in the PADC, will make it possible to correlate physical dose with direct cellular effects. This is a promising method for single cell analysis and the study of the effects of ionizing radiation at low particle flux density.     

Immunohistochemical expression of rabphilin-3A-like (Noc2) in normal and tumor tissues of human endocrine pancreas

Involvement of rabphilin-3A-like (RPH3AL), or Noc2, the potential effector of Ras-associated binding proteins Rab3A and Rab27A in the regulation of exocytotic processes in the endocrine pancreas has been demonstrated in experimental models. Noc2 expression together with other regulatory molecules of the exocytotic machinery in human tissues, however, has not been studied. We evaluated immunohistochemical expression of the key molecules of the exocytotic machinery, Noc2, Rab3A, Rab27A, and RIM2, together with the characteristic islet cell hormones, insulin and glucagon in normal and endocrine tumor tissues of human pancreas. Normal pancreatic islets were stained for all of these proteins and showed strong cytoplasmic localization. A similar pattern of strong cytoplasmic expression of these proteins was observed in the majority of endocrine tumors. By contrast, the exocrine portions of normal appearing pancreas completely lacked Rab27A staining and showed decreased expression of the proteins, Noc2, Rab3A, and RIM2. The staining pattern of Noc2 and Rab27A was similar to the staining pattern of glucagon-producing cells within the islets. The concomitant expression of Noc2 with these molecules suggests that Noc2 may serve as an effector for Rab3A and Rab27A and that it is involved in the regulation of exocytosis of the endocrine pancreas in humans.     

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular basis of recognition by Gal/GalNAc specific legume lectins: influence of Glu 129 on the specificity of peanut agglutinin (PNA) towards C2-substituents of galactose

The ability to discriminate between galactose and N- acetylgalactosamine, observed in some lectins, is crucial for their biological activity as well as their usefulness as tools in biology and medicine. However, the molecular basis of differential binding of lectins to these two sugars is poorly understood. Peanut agglutinin (PNA) is one of the few galactose-specific legume lectins which does not bind N- acetylgalactosamine at all and is, therefore, ideal for the study of the basis of specificity towards C-2 substituted derivatives of galactopyranosides. Examination of the three-dimensional structure of PNA in complex with lactose revealed the presence of both a longer loop and bulkier residues in the region surrounding the C-2 hydroxyl of the galactopyranoside ring, which can sterically prevent the accommodation of a bulky substituent in this position. One such residue, is a glutamic acid at position 129 which protrudes into the binding site and perhaps directly obstructs any substitution at the C-2 position. Two mutants in bacterially expressed PNA were therefore constructed. These were E129D and E129A, in which Glu129 was replaced by Asp and Ala, respectively. The specificity of the mutants for galactose, galactosamine, and N- acetylgalactosamine was examined through observing the inhibition of hemagglutination and binding of the lectin to immobilized asialofetuin. The results showed that the affinity of E129A and E129D for C-2-substituted derivatives of the galactose varies. The mutant E129D showed significant binding towards N- acetylgalactosamine, suggesting that the residue Glu 129 is crucial in imparting exclusive galactose-specificity upon PNA. This study not only attempts to provide an explanation for the inability of PNA to accommodate C-2-substituted derivatives at its primary subsite, but also seeks to present a basis for engineering lectins with altered specificities.      

Phosphorus mining for ecological restoration on former agricultural land

To restore species‐rich terrestrial ecosystems on ex‐agricultural land, establishing nutrient limitation for dominant plant growth is essential because in nutrient‐rich soils, fast‐growing species often exclude target species. However, N‐limitation is easier to achieve than P‐limitation (because of a difference in biogeochemical behavior), biodiversity is generally highest under P‐limitation. Commonly used restoration methods to achieve low soil P‐concentrations are either very expensive or take a very long time. A promising restoration technique is P‐mining, an adjusted agricultural technique that aims at depleting soil‐P. High biomass production and hence high P‐removal with biomass are obtained by fertilizing with nutrients other than P. A pot experiment was set up to study P‐mining with Lolium perenne L. on sandy soils with varying P‐concentrations: from an intensively used agricultural soil to a soil near the soil P‐target for species‐rich Nardus grassland. All pots received N‐ and K‐fertilization. The effects of biostimulants on P‐uptake were also assessed by the addition of arbuscular mycorrhiza (Glomus spp.), humic substances or phosphate‐solubilizing bacteria (Bacillus sp. and Pseudomonas spp.). In our P‐rich soil (111 µg P_Olsen/g), P‐removal rate was high but bioavailable soil‐P did not decrease. At lower soil P‐concentrations (64 and 36 µg P_Olsen/g), bioavailable soil‐P had decreased but the P‐removal rate had by then dropped 60% despite N‐ and K‐fertilization and despite that the target (<10 µg P_Olsen/g) was still far away. None of the biostimulants altered this trajectory. Therefore, restoration will still take decades when starting with ex‐agricultural soils unless P‐fertilization history was much lower than average.     

Phosphorylated BRCA1 is predominantly located in the nucleus and mitochondria

Multiple copies of the mitochondrial genome in eukaryotic cells are organized into protein-DNA complexes called nucleoids. Mitochondrial genome repair mechanisms have been reported, but they are less well characterized than their nuclear counterparts. To expand our knowledge of mitochondrial genome maintenance, we have studied the localization of the BRCA1 protein, known to be involved in nuclear repair pathways. Our confocal and immunoelectron microscopy results show that BRCA1 is present in mitochondria of several human cancer cell lines and in primary breast and nasal epithelial cells. BRCA1 localization in mitochondria frequently overlapped that of nucleoids. Small interfering RNA-mediated knockdown of BRCA1 in human cancer cells (confirmed by Western blot) results in decreased nuclear, cytoplasmic, and mitochondrial staining after immunofluorescence microscopy, establishing the specificity of the BRCA1 immunolabeling. Furthermore, using cell fractionation, dephosphorylation, and enzyme protection experiments, we show that a 220-kDa phosphorylated isoform of BRCA1 is enriched in mitochondrial and nuclear fractions but reduced in cytoplasmic subcellular fractions. Submitochondrial fractionation confirmed the presence of BRCA1 protein in isolated mitoplasts. Because phosphorylation of BRCA1 and subsequent changes in subcellular localization are known to follow DNA damage, our data support a universal role for BRCA1 in the maintenance of genome integrity in both mitochondria and nucleus.