The interaction between vitamin D receptor polymorphisms and sun exposure around time of diagnosis influences melanoma survival

Evidence on the relationship between the vitamin D pathway and outcomes in melanoma is growing, although it is not always clear. We investigated the impact of measured levels of sun exposure at diagnosis on associations of vitamin D receptor gene (VDR) polymorphisms and melanoma death in 3336 incident primary melanoma cases. Interactions between six SNPs and a common 3′‐end haplotype were significant (p < .05). These SNPs, and a haplotype, had a statistically significant association with survival among subjects exposed to high UVB in multivariable regression models and exerted their effect in the opposite direction among those with low UVB. SNPs rs1544410/BsmI and rs731236/TaqI remained significant after adjustment for multiple testing. These results suggest that the association between VDR and melanoma‐specific survival is modified by sun exposure around diagnosis, and require validation in an independent study. Whether the observed effects are dependent or independent of vitamin D activation remains to be determined.     

The balance between different peptidoglycan precursors determines whether Escherichia coli cells will elongate or divide.

The rodA(Sui) mutation allows cell division to take place at 42 degrees C in ftsI23 mutant cells, which produce a thermolabile penicillin-binding protein 3 (PBP3, the septation-specific peptidoglycan transpeptidase). We show here that the mutation in rodA is a single-base change from a glutamine to a chain termination (amber) codon, and that an amber suppressor (supE) present in the strain restores the ability to produce a reduced level of normal RodA protein. The reduced level of RodA is accompanied by an increase in the levels of two other proteins (PBP2 and PBP5) encoded by genes in the rodA operon. We show that an increased level of PBP5 is by itself sufficient to restore cell division to ftsI23 cells at 42 degrees C. Two other treatments were found to restore division capacity to the mutant: an increase in PBP6 (which is a D-alanine carboxypeptidase like PBP5) or suitable concentrations of D-cycloserine. All of the above treatments have the effect of reducing the number of pentapeptide side chains in peptidoglycan and increasing the number of tripeptides. We conclude that the effect of the rodA(Sui) mutation is to indirectly increase the availability of tripeptide side chains, which are used preferentially by PBP3 as acceptors in transpeptidation. A change in the proportions of different kinds of peptide side chain in the peptidoglycan can therefore determine whether cells will divide.     

Statistical tests for clonality

Cancer investigators frequently conduct studies to examine tumor samples from pairs of apparently independent primary tumors with a view to determine whether they share a "clonal" origin. The genetic fingerprints of the tumors are compared using a panel of markers, often representing loss of heterozygosity (LOH) at distinct genetic loci. In this article we evaluate candidate significance tests for this purpose. The relevant information is derived from the observed correlation of the tumors with respect to the occurrence of LOH at individual loci, a phenomenon that can be evaluated using Fisher's exact test. Information is also available from the extent to which losses at the same locus occur on the same parental allele. Data from these combined sources of information can be evaluated using a simple adaptation of Fisher's exact test. The test statistic is the total number of loci at which concordant mutations occur on the same parental allele, with higher values providing more evidence in favor of a clonal origin for the two tumors. The test is shown to have high power for detecting clonality for plausible models of the alternative (clonal) hypothesis, and for reasonable numbers of informative loci, preferably located on distinct chromosomal arms. The method is illustrated using studies to identify clonality in contralateral breast cancer. Interpretation of the results of these tests requires caution due to simplifying assumptions regarding the possible variability in mutation probabilities between loci, and possible imbalances in the mutation probabilities between parental alleles. Nonetheless, we conclude that the method represents a simple, powerful strategy for distinguishing independent tumors from those of clonal origin.     

A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo

Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to calsequestrin, the well-characterized Ca++ storage protein in the sarcoplasmic reticulum of muscle cells. In semi-thin frozen sections of unfertilized eggs, immunofluorescent staining revealed a tubuloreticular network throughout the cytoplasm. Staining of isolated egg cortices with the CSL protein antiserum showed the presence of a submembranous polygonal, tubular network similar to ER network patterns seen in other cells and in egg cortices treated with the membrane staining dye DiIC16[3]. In frozen sections of embryos during interphase of the first cell cycle, a cytoplasmic network similar to that of the unfertilized egg was present. During mitosis, we observed a dramatic concentration of the antibody staining within the asters of the mitotic apparatus where ER is known to aggregate. Electron microscopic localization on unfertilized eggs using peroxidase-labeled secondary antibody demonstrated the presence of the CSL protein within the luminal compartment of ER-like tubules. Finally, in frozen sections of centrifugally stratified eggs, the immunofluorescent staining concentrated in the clear zone: a layer highly enriched in ER and thought to be the site of calcium release upon fertilization. This localization of a CSL protein within the ER of the egg provides evidence for the ability of this organelle to serve a Ca++ storage role in the regulation of intracellular Ca++ in nonmuscle cells in general, and in the regulation of fertilization and cell division in sea urchin eggs in particular.     

Do Plant Cells Secrete Exosomes Derived from Multivesicular Bodies?

Multivesicular bodies (MVBs) are spherical endosomal organelles containing small vesicles formed by inward budding of the limiting membrane into the endosomal lumen. In mammalian red cells and cells of immune system, MVBs fuse with the plasma membrane in an exocytic manner, leading to release their contents including internal vesicles into the extracellular space. These released vesicles are termed exosomes. Transmission electron microscopy studies have shown that paramural vesicles situated between the plasma membrane and the cell wall occur in various cell wall-associated processes and are similar to exosomes both in location and in morphology. Our recent studies have revealed that MVBs and paramural vesicles proliferate when cell wall appositions are rapidly deposited beneath fungal penetration attempts or during plugging of plasmodesmata between hypersensitive cells and their intact neighboring cells. This indicates a potential secretion of exosome-like vesicles into the extracellular space by fusion of MVBs with the plasma membrane. This MVB-mediated secretion pathway was proposed on the basis of pioneer studies of MVBs and paramural vesicles in plants some forty years ago. Here, we recall the attention to the occurrence of MVB-mediated secretion of exosomes in plants.Key Words: cell wall, endocytosis, endosome, exocytosis, exosome, multivesicular body, paramural bodyMultivesicular bodies (MVBs) are spherical endosomal organelles containing a number of small vesicles formed by inward budding of the limiting membrane into the endosomal lumen.¹ MVBs contain endocytosed cargoes and deliver them into lysosomal/vacuolar compartments for degradation. They also incorporate newly synthesized proteins destined for lysosomal/vacuolar compartments.² In mammalian cells of hematopoietic origin, endosomal MVBs function in removal of endocytosed surface proteins in an exocytic manner. They are redirected to the plasma membrane, where they release their contents including internal vesicles into the extracellular space by membrane fusion. The released vesicles are termed exosomes.³ During reticulocyte maturation to erythrocyte, a group of surface proteins, such as the transferrin receptor, become obsolete and are discarded via MVB-mediated secretion.³ Time-course transmission electron microscopy (TEM) first revealed that colloidal gold-transferrin was internalized into MVBs via receptor-mediated endocytosis and then transferrin together with its receptor were delivered into the extracellular space via the fusion of MVBs with the plasma membrane of reticulocytes.⁴ Some other cell types of hematopoietic origin, such as activated platelets, cytotoxic T cells and antigen-presenting cells, also secrete exosomes. Exosomes thus may play a role in various physiological processes other than discarding obsolete proteins.³Our recent TEM studies provided ultrastructural evidence on the enhanced vesicle trafficking in barley leaf cells attacked by the biotrophic powdery mildew fungus. Multivesicular compartments including MVBs, intravacuolar MVBs, and paramural bodies turned out to proliferate in intact host cells during formation of cell wall appositions (papilla response), in the hypersensitive response, and during accommodation of haustoria.^5,6 MVBs proliferated in the cytoplasm of haustorium-containing epidermal cells during compatible interactions and near sites of cell wall-associated oxidative microburst either during the papilla response or during the hypersensitive response. Because MVBs in plant cells have been demonstrated to be endosomal compartments,^7–9 they may participate in internalization of nutrients from the apoplast of intact haustorium-containing epidermal cells and sequestration of damaged membranes and deleterious materials originating from the oxidative microburst.^5,6 The presence of intravacuolar MVBs with double limiting membranes (Fig. 1A) indicates an engulfment of MVBs by the tonoplast and a vacuole-mediated autophagy of MVBs.^5,6 MVBs, as prevacuolar compartments in plant cells,⁹ thus probably deliver their contents into the central vacuole via both the fusion with the tonoplast and the engulfment by the tonoplast (Fig. 2A and B). On the other hand, paramural bodies, in which small vesicles are situated between the cell wall and the plasma membrane, were associated with cell wall appositions deposited beneath fungal penetration attempts (Fig. 1B) or around hypersensitive cells including sites of plugged plasmodesmata (Fig. 1C and D).^5,6 Because paramural vesicles are similar to exosomes both in location and in morphology, we speculated that MVBs fuse with the plasma membrane in an exocytic manner to form paramural bodies.^5,6 Endocytosed cell surface materials in endosomal MVBs may be reused and delivered together with newly synthesized materials in Golgi apparatus-derived vesicles to cell wall appositions, which are deposited rapidly to prevent fungal penetration (Fig. 2A) or to contain hypersensitive cell death (Fig. 2B). MVBs thus may be driven along two distinct pathways to deliver their contents into either central vacuole or extracellular space.Open in a separate windowFigure 1Multivesicular compartments in intact cells in barley leaves attacked by the barley powdery mildew fungus. (A) An intravacuolar multivesicular body (MVB) with double limiting membranes in an intact epidermal cell (EC) adjacent to a hypersensitive epidermal cell (EC*). The arrows point to the outer limiting membrane, which is seemingly derived from the tonoplast. Note that neighboring intravacuolar vesicles (in between two arrowheads) may result from degradation of double limiting membranes of intravacuolar MVBs or may be delivered into the vacuole by MVB-fusion with the tonoplast. (B) Paramural vesicles (arrowheads) in a paramural body associated with cell wall appositions (asterisk) deposited by an intact epidermal cell. (C) A multivesicular body (MVB) in contact with a paramural body (PMB) (a nonmedian section) associated with cell wall appositions (asterisk) deposited by an intact mesophyll cell adjacent to a hypersensitive mesophyll cell. Note that cell wall appositions deposit beside an intercellular space (IS). The arrows point to the tonoplast. (D) A paramural body (PMB) associated with cell wall appositions (asterisks) blocking plasmodesmata (in between two arrowheads) at the side of an intact mesophyll cell (MC) underlying a hypersensitive epidermal cell (EC*). The arrows point to the tonoplast. CV, central vacuole; CW, cell wall; MB, microbody. Bars, 1µm.Open in a separate windowFigure 2Hypothetical diagram of delivery of endocytosed cell surface materials via MVBs into the central vacuole or the extracellular space where intact barley cells deposit cell wall appositions. (A) Deposition of cell wall appositions (asterisk) beneath powdery mildew penetration attempts. AGT, appressorial germ tube; PP, penetration peg. (B) Deposition of cell wall appositions (asterisks) against constricted plasmodesmata (PD) between a hypersensitive epidermal cell (EC) penetrated by the powdery mildew fungus and an underlying mesophyll cell (MC). H, haustorium. Arrows and numbers show pathways of vesicle trafficking. 1, Secretion of Golgi-derived vesicles containing newly synthesized materials; G, Golgi body; TGN, trans-Golgi network; 2, Endocytosis of cell surface materials from coated pits (coated open circles) via coated vesicles (coated circles) to multivesicular bodies (MVB); 3, Delivery of endocytosed materials for degradation inside the central vacuole (CV) via membrane fusion between MVBs and the tonoplast (T); small broken circles, vesicles in degradation; 4, Delivery of endocytosed materials for degradation inside the central vacuole via engulfment of MVBs by the tonoplast; large broken circles; MVB limiting membranes in degradation; 5, delivery of endocytosed materials into the extracellular space for deposition of cell wall appositions (asterisks) via membrane fusion between MVBs and the plasma membrane (PM). CW, cell wall; PMB, paramural body. PD0, 1, 2, 3 and 4 represent stages of plugging plasmodesmata. PD0, open plasmodesmata between two intact mesophyll cells (MC) subjacent to the hypersensitive epidermal cell (EC); PD1, constriction of plasmodesmata by callose (grey dots) deposition at plasmodesmal neck region; PD2, constricted plasmodesmata associated with plasmodesma-targeted secretion; PD3, further blocking of plasmodesmata by deposition of cell wall appositions; PD4, completely blocked plasmodesmata.Earlier than the discovery in animal cell systems,⁴ it was proposed in two independent papers in 1967 that the fusion of MVBs with the plasma membrane might result in the release of small vesicles into the extracellular space in fungi and in higher plants.^10,11 Several lines of evidence support the occurrence of MVB-mediated secretion of exosome-like vesicles in plants. First, vesicles of the same morphology as MVB internal vesicles have been observed in extracellular spaces or paramural spaces in various types of plant cells in various plant species by TEM.¹² An early study on endocytosis by soybean protoplasts also showed small extracellular vesicles attaching on the plasma membrane.⁸ Second, cooccurrence of MVBs and paramural vesicles has been observed in processes of cell proliferation, cell differentiation, and cell response to abiotic and biotic stress. Examples are cell plate formation,^13,14 secondary wall thickening,^15,16 cold hardness,^17,18 and deposition of cell wall appositions upon pathogen attack.^5,6,19–21 Third, identical molecular components, such as arabinogalactan proteins^22,23 and peroxidases,⁶ have been immunolocalized in both MVBs and paramural bodies. Despite these pieces of evidence, a conclusive demonstration of MVB-mediated secretion of exosomes in plants requires further exploration.The presently available experimental systems, approaches, and membrane markers may allow future demonstration of MVB-mediated secretion of exosomes in plants. Recent in vivo real-time observation and colocalization of cell surface and endosomal markers have already revealed that endosomes filled with endocytosed preexisting cell wall and plasma membrane materials are rapidly delivered to cytokinetic spaces to form cell plates in dividing tobacco, Arabidopsis, and maize cells.²⁴ Because TEM observed paramural bodies attaching to cell plates¹³ and MVBs in the vicinity of cell plates during all stages of cell plate formation,^14,25,26 MVBs and paramural bodies may participate in delivery of endocytosed building blocks to cell plates. Jiang''s and Robinson''s labs together developed a transgenic tobacco BY-2 cell line stably expressing a YFP-labeled vacuolar sorting receptor protein and antibodies against the vacuolar sorting receptor protein localized to the limiting membrane of MVBs.⁹ These tools together with live cell imaging and immunoelectron microscopy may allow visualization of MVB-fusion to the new plasma membrane, of vacuolar sorting receptors in both the limiting membrane of MVBs and the new plasma membrane, and of identical cell plate components in both internal vesicles of MVBs and paramural vesicles.In spite of obvious differences in plant and animal cytokinesis, the generation of cell plates by cell-plate-directed fusion of endosomes resembles the plugging of midbody canals by midbody-directed endosomes to separate daughter cells at the terminal phase of animal cytokinesis.²⁷ Likely, functional similarities of the fusion between endosomal MVBs and the plasma membrane to eliminate unwanted cell contents may also exist in maturation of mammalian red blood cells and plant sieve elements in the sense that the fusion of MVBs with the plasma membrane may occur during maturation of the latter.²⁸ On the other hand, although plant cells may secrete MVB-derived exosomes in defense response upon pathogen attack,^5,6 plant cell walls rule out the direct intercellular communication during the immune response mediated by exosomes in the circulation of mammals.³ In contrast, plasmodesma-directed secretion of exosomes would block the cell-to-cell communication between hypersensitive cells and their neighboring cells during hypersensitive response.⁵ Further exploration will lead us to a better understanding of similarities and differences of exosome secretion between plants and animals.     

Support for Sustainable Development Policy Decisions A Case Study from Highway Maintenance

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http://dx.doi.org/10.1065/lca2006.04.009

Goal, Scope and Method

logy. This paper describes a case study carried out as part of a wider programme to provide support for environmental decision-making in the highway maintenance programme of a local government body: Surrey County Council (SCC). UK local authorities are required to demonstrate that sustainable development principles are addressed in service provision, by improving environmental, economic or social wellbeing and improving public consultation. A methodological approach was developed to meet these requirements by using life cycle assessment (LCA) and multi-criteria decision analysis (MCDA) through the process of decision conferencing.

Results

In projects requiring strategic decisions, difficulties arise in identifying relevant sustainable development criteria and in evaluating maintenance options against these criteria where the context for decision-making is complex and characterised by uncertainty, where multiple public policy objectives compete and a number of decision-makers and key players are affected by the outcome. Clearly, a structured process is needed to engage such stakeholders in the decision process, utilising quantitative and qualitative information. The approach described proved to be capable of fulfilling these requirements.

Conclusions

and Recommendations. The approach of combining LCA with MCDA through decision conferencing is capable of further development to support other strategic decision-making activities. However, this illustrative case study has revealed a need for methodological developments in LCA for local, project-level decisions.

     

Two-stage designs for gene-disease association studies with sample size constraints

Gene-disease association studies based on case-control designs may often be used to identify candidate polymorphisms (markers) conferring disease risk. If a large number of markers are studied, genotyping all markers on all samples is inefficient in resource utilization. Here, we propose an alternative two-stage method to identify disease-susceptibility markers. In the first stage all markers are evaluated on a fraction of the available subjects. The most promising markers are then evaluated on the remaining individuals in Stage 2. This approach can be cost effective since markers unlikely to be associated with the disease can be eliminated in the first stage. Using simulations we show that, when the markers are independent and when they are correlated, the two-stage approach provides a substantial reduction in the total number of marker evaluations for a minimal loss of power. The power of the two-stage approach is evaluated when a single marker is associated with the disease, and in the presence of multiple disease-susceptibility markers. As a general guideline, the simulations over a wide range of parametric configurations indicate that evaluating all the markers on 50% of the individuals in Stage 1 and evaluating the most promising 10% of the markers on the remaining individuals in Stage 2 provides near-optimal power while resulting in a 45% decrease in the total number of marker evaluations.     

Rapid and simple high-performance liquid chromatographic assay for the determination of metformin in human plasma and breast milk

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of metformin in human plasma and breast milk is described. After proteins were precipitated with acetonitrile, metformin and the internal standard buformin were resolved on a cation-exchange column and detected by UV detection at 236 nm. Standard curves were linear over the concentration range 20.0-4000 microg/l. Intra- and inter-day coefficients of variation were <9.0% and the limit of quantification was around 20 microg/l.     

Genetic variation and stock structure of school mackerel and spotted mackerel in northern Australian waters

The total mean sample heterozygosity calculated from eight polymorphic loci was 0·172 (0·047 S.E.) (F_st 0·025) for school mackerel Scomberomorus queenslandicus , and for spotted mackerel S. munroi was 0·110 (0·074 S.E.) (F_st 0·038). There was no evidence of temporal variation for either species as significant genetic differences were not detected between months or year classes within areas. Spatially, school mackerel have a complex stock structure, with stocks being associated with large embayments. In contrast, spotted mackerel appear to comprise a single stock in Australian east coast waters. Both species showed a significant pattern of stock structure between Australian east coast and northern (Arafura Sea) samples.     

The chemistry of the lowland rice rhizosphere

Models and experimental studies of the rhizosphere of rice plants growing in anaerobic soil show that two major processes lead to considerable acidification (1–2 pH units) of the rhizosphere over a wide range of root and soil conditions. One is generation of H⁺ in the oxidation of ferrous iron by O₂ released from the roots. The other is release of H⁺ from roots to balance excess intake of cations over anions, N being taken up chiefly as NH₄ ⁺. CO₂ exchange between the roots and soil has a much smaller effect. The zone of root-influence extends a few mm from the root surface. There are substantial differences along the root length and with time. The acidification and oxidation cause increased sorption of NH₄ ⁺ ions on soil solids, thereby impeding the movement of N to absorbing root surfaces. But they also cause solubilization and enhanced uptake of soil phosphate.