Development and maturation of surghum seeds on detached panicles grown in vitro

Summary A procedure for culturing detached panicles of sorghum, Sorghum bicolor (L.) Moench, was developed to achieve flowering, fertilization, and subsequent seed development and maturation in vitro. Sixteen sorghum genotypes (five high and eleven low in tannin) were tested for their ability to develop normally in culture. Panicles collected one to two days before the initiation of anthesis were cultured in flasks containing liquid medium. Contamination and medium darkening were the major obstacles encountered. Up to 55% of the panicles cultured reached physiological maturity in vitro. The frequency of seed set ranged from 30 to 97% depending upon genotype and medium. Seed and glume color were normal. Seed produced in vitro resembled those grown in vivo and germinated well, but were smaller than normal (100 kernel weight reached 50 to 70% of the control). Grain polyphenols were synthesized in the cultured panicles. Seed of high tannin genotypes produced in vitro were lower in total phenols and tannins and higher in flavan-4-ols and the 3-deoxyanthocyanidin pigments than control seed. This technique can be used for harvesting late-maturing stocks and for various sorghum studies.     

Intracellular aminopeptidases inStreptomyces lividans 66

Summary We have investigated the aminopeptidase activities present inStreptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues. Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels. One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis. The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.     

Direct visualization of the structure of the "20 S" aggregate of coat protein of tobacco mosaic virus. The "disk" is the major structure at pH 7.0 and the Proto-helix at lower pH.

We have employed the rapid-freeze technique to prepare specimens for electron microscopy of a coat protein solution of tobacco mosaic virus at equilibrium at pH 7.0 and 6.8, ionic strength 0.1 M and 20 degrees C. The former are the conditions for the most rapid assembly of the virus from its isolated protein and RNA. At both pH values, the equilibrium mixture contains approximately 80% of a "20 S" aggregate and 20% of a "4 S" aggregate (the so-called A-protein). The specimens were prepared either totally unstained or positively stained with methyl mercury nitrate, which binds to an amino acid residue (Cys27) internally located within the subunit, which we show not to affect the virus assembly. The images in the electron microscope are compatible only with the major structure for the "20 S" aggregate at pH 7.0 containing two rings of subunits and these aggregates display the same binding contacts as those seen between the aggregate that forms the asymmetric unit in the crystal, which has been shown by X-ray crystallography to be a disk containing two rings, each of 17 subunits, oriented in the same direction. In contrast, the images from specimens prepared at pH 6.8 show the major structure to be a proto-helix at this slightly lower pH, demonstrating that the technique of cryo-electron microscopy is capable of distinguishing between these aggregates of tobacco mosaic virus coat protein. The main structure in solution at pH 7.0 must therefore be very similar to that in the crystal, although slight differences could occur and there are probably other, minor, components in a mixture of species sedimenting around 20 S under these conditions. The equilibrium between aggregates is extremely sensitive to conditions, with a drop of 0.2 pH unit tipping the disk to proto-helix ratio from approximately 10:1 at pH 7.0 to 1:10 at pH 6.8. This direct determination of the structure of the "20 S" aggregate in solution, under conditions for virus assembly, contradicts some recent speculation that it must be helical, and establishes that, at pH 7.0, it is in fact predominantly a two-layer disk as it had been modelled before.     

FireStem2D – A Two-Dimensional Heat Transfer Model for Simulating Tree Stem Injury in Fires

FireStem2D, a software tool for predicting tree stem heating and injury in forest fires, is a physically-based, two-dimensional model of stem thermodynamics that results from heating at the bark surface. It builds on an earlier one-dimensional model (FireStem) and provides improved capabilities for predicting fire-induced mortality and injury before a fire occurs by resolving stem moisture loss, temperatures through the stem, degree of bark charring, and necrotic depth around the stem. We present the results of numerical parameterization and model evaluation experiments for FireStem2D that simulate laboratory stem-heating experiments of 52 tree sections from 25 trees. We also conducted a set of virtual sensitivity analysis experiments to test the effects of unevenness of heating around the stem and with aboveground height using data from two studies: a low-intensity surface fire and a more intense crown fire. The model allows for improved understanding and prediction of the effects of wildland fire on injury and mortality of trees of different species and sizes.     

Thinking and managing outside the box: coalescing connectivity networks to build region-wide resilience in coral reef ecosystems

As the science of connectivity evolves, so too must the management of coral reefs. It is now clear that the spatial scale of disturbances to coral reef ecosystems is larger and the scale of larval connectivity is smaller than previously thought. This poses a challenge to the current focus of coral reef management, which often centers on the establishment of no-take reserves (NTRs) that in practice are often too small, scattered, or have low stakeholder compliance. Fished species are generally larger and more abundant in protected reserves, where their reproductive potential is often greater, yet documented demographic benefits of these reproductive gains outside reserves are modest at best. Small reproductive populations and limited dispersal of larvae play a role, as does the diminished receptivity to settling larvae of degraded habitats that can limit recruitment by more than 50%. For “demographic connectivity” to contribute to the resilience of coral reefs, it must function beyond the box of no-take reserves. Specifically, it must improve nursery habitats on or near reefs and enhance the reproductive output of ecologically important species throughout coral reef ecosystems. Special protection of ecologically important species (e.g., some herbivores in the Caribbean) and size-regulated fisheries that capitalize on the benefits of NTRs and maintain critical ecological functions are examples of measures that coalesce marine reserve effects and improve the resilience of coral reef ecosystems. Important too is the necessity of local involvement in the management process so that social costs and benefits are properly assessed, compliance increased and success stories accrued.     

Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Time-dependent botulinum neurotoxin serotype A metalloprotease inhibitors

Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes, the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa, is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure–activity relationships and a proposed mechanism of action for this novel inhibitor chemotype.     

RHODELLA RETICULATA SP. NOV., A NEW COCCOID RHODOPHYTAN ALGA (PORPHYRIDIALES)1,2

A new coccoid rhodophytan species is described and compared with other species in the genus Rhodella. Thylakoids in the pyrenoid characterize the new species and indicate a closer relationships of Rhodella to Porphyridium than was previously indicated.