Structural investigations on betacyanin pigments by LC NMR and 2D NMR spectroscopy

Four betacyanin pigments were analysed by LC NMR and subjected to extensive NMR characterisation after isolation. Previously, low pH values were applied for NMR investigations of betalains resulting in rapid degradation of the purified substances thus preventing extensive NMR studies. Consequently, up to now only one single (13)C NMR spectrum of a betalain pigment, namely that of neobetanin (=14,15-dehydrobetanin), was available. Because of its sufficient stability under highly acidic conditions otherwise detrimental for betacyanins, this pigment remained an exemption. Since betalains are most stable in the pH range of 5-7, a new solvent system has been developed allowing improved data acquisition through improved pigment stability at near neutral pH. Thus, not only (1)H, but for the first time also partial (13)C data of betanin, isobetanin, phyllocactin and hylocerenin isolated from red-purple pitaya [Hylocereus polyrhizus (Weber) Britton & Rose, Cactaceae] could be indirectly obtained by gHSQC- and gHMQC-NMR experiments.     

Phycobilisome diffusion is required for light-state transitions in cyanobacteria

Phycobilisomes are the major accessory light-harvesting complexes of cyanobacteria and red algae. Studies using fluorescence recovery after photobleaching on cyanobacteria in vivo have shown that the phycobilisomes are mobile complexes that rapidly diffuse on the thylakoid membrane surface. By contrast, the PSII core complexes are completely immobile. This indicates that the association of phycobilisomes with reaction centers must be transient and unstable. Here, we show that when cells of the cyanobacterium Synechococcus sp. PCC7942 are immersed in buffers of high osmotic strength, the diffusion coefficient for the phycobilisomes is greatly decreased. This suggests that the interaction between phycobilisomes and reaction centers becomes much less transient under these conditions. We discuss the possible reasons for this. State transitions are a rapid physiological adaptation mechanism that regulates the way in which absorbed light energy is distributed between PSI and PSII. Immersing cells in high osmotic strength buffers inhibits state transitions by locking cells into whichever state they were in prior to addition of the buffer. The effect on state transitions is induced at the same buffer concentrations as the effect on phycobilisome diffusion. This implies that phycobilisome diffusion is required for state transitions. The main physiological role for phycobilisome mobility may be to allow such flexibility in light harvesting.     

Untreated silicone breast implant rupture

Implant rupture is a well-known complication of breast implant surgery that can pass unnoticed by both patient and physician. To date, no prospective study has addressed the possible health implications of silicone breast implant rupture. The aim of the present study was to evaluate whether untreated ruptures are associated with changes over time in magnetic resonance imaging findings, serologic markers, or self-reported breast symptoms. A baseline magnetic resonance imaging examination was performed in 1999 on 271 women who were randomly chosen from a larger cohort of women having cosmetic breast implants for a median period of 12 years (range, 3 to 25 years). A follow-up magnetic resonance imaging examination was carried out in 2001, excluding women who underwent explantation in the period between the two magnetic resonance imaging examinations (n = 44). On the basis of these examinations, the authors identified 64 women who had at least one ruptured implant at the first magnetic resonance imaging examination and, for comparison, all women who had intact implants at both examinations (n = 98). Magnetic resonance images from the two examinations were compared and changes in rupture configuration were evaluated. Comparisons were also made for self-reported breast symptoms occurring during the study period and for changes in serum values of antinuclear antibodies, rheumatoid factor, and cardiolipin antibodies immunoglobulin G and immunoglobulin M. The majority of the women with implant rupture had no visible magnetic resonance imaging changes of their ruptured implants. For 11 implants (11 percent) in 10 women, the authors observed progression of silicone seepage, either as a conversion from intracapsular into extracapsular rupture (n = 7), as progression of extra-capsular silicone (n = 3), or as increasing herniation of the silicone within the fibrous capsule (n = 1); however, in most cases, these changes were minor. Some changes could be ascribed to trauma, but others seemed spontaneous. There was no increase in levels of autoantibodies during the study period in either study group. Women with untreated implant ruptures reported a significant increase in nonspecific breast changes (odds ratio, 2.1; 95 percent confidence interval, 1.2 to 3.8) compared with women without ruptures. On the basis of this first study of women with untreated silicone breast implant rupture, the authors conclude that implant rupture is a relatively harmless condition, which only rarely progresses and gives rise to notable symptoms. Even so, because of a small risk of silicone spread, the authors suggest that women with implant ruptures be followed clinically, if not operated on. Because implant ruptures often occur asymptomatically, any woman with silicone implants, regardless of rupture status, should be evaluated at regular intervals.     

Southern Sea Otter as a Sentinel of Marine Ecosystem Health

The southern sea otter (Enhydra lutris nereis) is listed as threatened under the Endangered Species Act (ESA) and is a keystone species, strongly influencing the abundance and diversity of the other species within its kelp forest ecosystem. This is accomplished primarily by preying upon urchins that eat the kelp stipe and holdfast, which can reduce a kelp forest to an urchin barren. Sea otters are very susceptible to marine pollutants such as petroleum, which may be directly toxic and/or alter their furs insulating properties. Sea otters are an excellent sentinel species. They eat approximately 25% of their body weight per day in shellfish and other invertebrates, and can concentrate and integrate chemical contaminants. In addition, they appear to be susceptible to a number of diseases and parasites that may have anthropogenic origins, and shellfish may serve as an intermediary for some of these infections. Many of the shellfish the otters eat are also harvested for human food. In their role as sentinels, sea otter health has implications for human health, economic sustainability of shellfisheries, as well as overall marine ecosystem health. The recent southern sea otter decline has been viewed with some alarm by conservationists and, indeed, recovery seems a long way off. High mortality rather than depressed recruitment appears to underlie the decline. A good deal of debate has centered on the role of infectious diseases and parasites, exposure to contaminants, nutrition and prey availability, net and pot fishery interactions, and other sources of mortality. Current research is being done related to major classes of mortality, various types of pollutants and some specific organisms causing southern sea otter mortality, and their implications for marine ecosystem health and sustainability.     

Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3

The Bacillus subtilis strain A1/3 shows exceptionally diverse antibiotic capacities compared to other B. subtilis strains. To analyze this phenomenon, mutants for the putative pantotheinyltransferase gene ( pptS), and for several genes involved in non-ribosomal peptide synthesis and polyketide synthesis were constructed and characterized, using bioassays with blood cells, bacterial and fungal cells, and mass spectrometry. Among at least nine distinct bioactive compounds, five antibiotics and one siderophore activity were identified. The anti-fungal and hemolytic activities of strain A1/3 could be eliminated by mutation of the fen and srf genes essential for the synthesis of fengycins and surfactins. Both pptS - and dhb -type mutants were defective in iron uptake, indicating an inability to produce a 2,3-dihydroxybenzoate-type iron siderophore. Transposon mutants in the malonyl CoA transacylase gene resulted in the loss of hemolytic and anti-fungal activities due to the inhibition of bacillomycin L synthesis, and this led to the discovery of bmyLD-LA-LB* genes. In mutants bearing disruption mutations in polyketide ( pksM - and/or pksR -like) genes, the biosynthesis of bacillaene and difficidins, respectively, was inactivated and was accompanied by the loss of discrete antibacterial activities. The formation of biofilms (pellicles) was shown to require the production of surfactins, but no other lipopeptides, indicating that surfactins serve specific developmental functions.Communicated by A. Kondorosi     

Neogenin mediates the action of repulsive guidance molecule

Repulsive guidance molecule (RGM) is a recently identified protein implicated in both axonal guidance and neural tube closure. The avoidance of chick RGM in the posterior optic tectum by growing temporal, but not nasal, retinal ganglion cell axons is thought to contribute to visual map formation. In contrast to ephrins, semaphorins, netrins and slits, no receptor mechanism for RGM action has been defined. Here, an expression cloning strategy identified neogenin as a binding site for RGM, with a sub-nanomolar affinity. Consistent with selective axonal responsiveness to RGM, neogenin is expressed in a gradient across the chick retina. Neogenin is known to be one of several netrin-binding proteins but only neogenin interacts with RGM. The avoidance of RGM by temporal retinal axons is blocked by the anti-neogenin antibody and the soluble neogenin ectodomain. Dorsal root ganglion axons are unresponsive to RGM but are converted to a responsive state by neogenin expression. Thus, neogenin functions as an RGM receptor.     

Patterning Axonal Guidance Molecules Using a Novel Strategy for Microcontact Printing

We present here a two-step strategy for micropatterning proteins on a substrate to control neurite growth in culture. First, conventional microcontact printing is used to prepare a micropattern of protein A, which binds the Fc fragment of immunoglobulins. Then, a chimeric protein, consisting of the extracellular domain of a guidance protein recombinantly linked to the Fc fragment of IgG (prepared using conventional molecular techniques), is applied from solution. The chimeric protein binds to the patterned protein A, taking on its geometric pattern. Using this method, we have micropatterned the extracellular domain of the cell adhesion molecule, L1 (as an L1-Fc chimera) and demonstrated that it retains its ability to selectively guide axonal growth. L1-Fc micropatterned on a background of poly-l-lysine resulted in selective growth of the axons on the micropattern, whereas the somata and dendrites were unresponsive. Substrates bearing simultaneous micropatterns of L1-Fc and poly-l-lysine on a background of untreated glass were also created. Using this approach, cell body position was controlled by manipulating the dimensions of the poly-l-lysine pattern, and the dendrites were constrained to the poly-l-lysine pattern, while the axons grew preferentially on L1-Fc. The two-step microcontact printing method allows preparation of substrates that contain guidance proteins in geometric patterns with resolution of 1 m. This method should be broadly applicable to many classes of proteins.     

Molecular interactions between an insect predator and its herbivore prey on transgenic potato expressing a cysteine proteinase inhibitor from rice

Transgenic plants expressing resistance to herbivorous insects may represent a safe and sustainable pest control alternative if they do not interfere with the natural enemies of target pests. Here we examined interactions between oryzacystatin I (OCI), a proteinase inhibitor from rice genetically engineered into potato (Solanum tuberosum cv. Kennebec, line K52) to increase resistance to insect herbivory, and the insect predator Perillus bioculatus. This stinkbug is a relatively specialized predator of caterpillars and leaf-beetle larvae, and may also include plant sap in its predominantly carnivorous diet. One of its preferred prey is Colorado potato beetle (Leptinotarsa decemlineata), a major target of insect resistance development for potato field crops. Gelatin/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that a major fraction of proteinase (gelatinase) activity in P. bioculatus extracts is OCI-sensitive. Among five gelatinolytic bands detected, the slowest-moving one (proteinase I) was inhibited strongly by purified OCI expressed in Escherichia coli or by OCI-transgenic potato extracts, while three other proteinases were partly sensitive to these treatments. There was also evidence of slight inhibition of proteinase I by untransformed potato foliage, suggesting the presence of a natural inhibitor related to OCI at low level in potato foliage. Interestingly, only about 50% of the maximum potential activity of proteinase I was recovered in extracts of P. bioculatus feeding on L. decemlineata larval prey on a diet of OCI-potato foliage, indicating that the predator was sensitive to OCI in the midgut of its prey. However, P. bioculatus on OCI-prey survived, grew and developed normally, indicating ability to compensate prey-mediated exposure to the OCI inhibitor. Confinement of P. bioculatus to potato foliage provided no evidence that potato plant-derived nutrition is a viable alternative to predation, restriction to potato foliage in fact being inferior to free water for short-term survival of nonfeeding first-instar larvae. These results support the view that OCI, an effective inhibitor of a substantial fraction of digestive enzymatic potential in P. bioculatus, should not interfere with its predation potential when expressed in potato plants fed to its prey at a maximum level of approximately 0.8% of total soluble proteins in mature foliage.     

Lipid diffusion in the thylakoid membranes of the cyanobacterium Synechococcus sp.: effect of fatty acid desaturation

Thylakoid membranes are crucial to photosynthesis in cyanobacteria and plants. In cyanobacteria, genetic modification of membrane lipid composition strongly influences cold tolerance and susceptibility to photoinhibition. We have used fluorescence recovery after photobleaching to measure the diffusion of a lipid-soluble fluorescent marker in cells of the cyanobacterium Synechococcus sp. PCC 7942. We have compared the wild-type strain with a transformant with an increased level of fatty acid unsaturation. The transformant showed a six-fold increase in the diffusion coefficient for the fluorescent marker at growth temperature. This is the first direct measurement of lipid diffusion in a photosynthetic membrane.     

High levels of extra-pair paternity in an isolated, low-density, island population of tree swallows (Tachycineta bicolor)

Molecular genetic studies have suggested that apparently nonbreeding males ('floaters') may account for a significant proportion of extra-pair paternity (EPP) in avian populations. Attempts to determine the influence of breeding density on EPP are therefore confounded by the presence of a subpopulation of floaters whose numbers are difficult to estimate. To study EPP in a tree swallow (Tachycineta bicolor) population with few floaters, we chose a nestbox grid on an island with an excess of available breeding sites and very few floaters. We obtained DNA samples from 13 complete families and performed DNA profiling on them using four microsatellite loci. For comparison, we also obtained a sample of 58 extra-pair young (EPY) from a mainland population typed at five microsatellite loci. Paternity assignments among resident males in both populations were made using the microsatellite profiles and a likelihood-based statistical method. Of the 67 island nestlings typed, we found 37 (55%) nestlings from 11 (85%) different nests that were EPY. The proportion of nestlings that were EPY and the proportion of broods containing EPY did not differ significantly between island and mainland populations studied previously. There was no significant difference between island and mainland populations in the proportion of extra-pair paternities assigned among neighbouring resident males. Male breeding density does not appear to affect the ability of female tree swallows to obtain extra-pair fertilizations, at least over the range of densities studied so far. The rate of EPP has remained remarkably consistent over many years, studies and populations implying an important role of active female choice in determining EPP.