The effect of caffeine,different fixation regimes and low temperature on microtubules in the cells of higher plants

Caffeine, (1:3:7-tri-methyl-xanthine), either as a prefixation treatment or included with glutaralde-hyde as the primary fixative, destroys or disorganises the microtubules associated with the formation of secondary walls in fibres from the flowering stem of the grass Lolium temulentum L. There is no observable effect of caffeine treatment on the microtubules associated with primary wall formation in collenchyma and young fibres from L. temulentum or in root cap cells of Zea mays L. and Phaseolus vulgaris L. The microtubules associated with primary wall formation are destroyed by cold treatment but not those associated with secondary wall formation. Tannic acid included in the fixative shows the microtubules associated with secondary wall formation in fibres of L. temulentum to be composed of 13 subunits. Treatment with lanthanum hydroxide does not stain the core or the halo of the microtubules.Abbreviation PIPES Piperazine N-N- bis 2 ethanol sulphonic acid The Grassland Research Institute is financed through the Agricultural Research Council     

Total lung capacity does not change during methacholine-stimulated airway narrowing

The accurate measurement of changes in flow rates from partial flow-volume curves depends on their measurement at the same lung volume. This lung volume can be standardized from total lung capacity (TLC) if this does not change at the same time. We examined the effect of methacholine-stimulated maximal airway narrowing [change in mean forced expiratory volume in 1 s (delta FEV1) = 26.4%] on TLC, measured by whole-body plethysmography, in 10 normal subjects and of moderate airway narrowing (mean delta FEV1 = 34.9%) in 10 asthmatics. The TLC changed from 5.88 to 6.03 liters in normal subjects (P greater than 0.05) and from 6.92 to 6.95 liters (P greater than 0.5) in asthmatics. The results of this study suggest that TLC does not change significantly after methacholine-stimulated maximal airway narrowing in normal subjects and after moderate narrowing in asthmatics.     

Butyric acid causes morphological changes in cultured chondrocytes through alterations in the extracellular matrix

Butyric acid induces characteristic changes in the morphology of chick embryo chondrocytes. Chick embryo chondrocytes when cultured in the absence of butyrate exhibit a spherical morphology and synthesize cartilage-specific chondroitin sulfate proteoglycan (CSPG). When these cultures are initiated and maintained in the presence of butyric acid, chondrocytes exhibit a mesenchymal morphology, a 90% reduction in the synthesis of CSPG, and a 75% reduction in DNA synthesis. The reduced synthesis of CSPG and DNA was shown not to be dependent on the morphological change. Chondrocytes require CSPG in order to express a spherical morphology, since including chondroitinase ABC in the culture media caused the cells to spread. In addition, the treatment of chondrocytes with purified CSPG prior to culture in media containing butyric acid resulted in spherical cells. The butyrate-induced spreading was shown to require either serum or fibronectin and could be prevented with antiserum against chick cell-surface fibronectin (cFn). Cell-surface fibronectin, which was present on both spherical and flattened chondrocytes, organized into fibrils beneath cells which spread. Increased fibronectin synthesis was not responsible for the butyrate-induced morphological change. From this evidence, it is concluded that the mechanism by which butyrate alters the morphology of these cells in culture involves inhibiting CSPG synthesis, thus preventing CSPG accumulation in the extracellular matrix (ECM). The absence of CSPG in the ECM allows fibronectin to mediate spreading of chondrocytes in culture.     

Inhibition of limb chondrogenesis in vitro by vitamin A: alterations in cell surface characteristics

Mesenchyme cells derived from embryonic mouse limb buds were cultured at high cell density. During the first 24 h in culture, groups of mesenchyme cells condensed and formed cell contacts and specialized junctions. These condensations were the nodule primordia which gave rise to cartilage nodules. The cell contacts were lost as the mesenchyme cells in the primordia developed into cartilage nodules. The mature nodules contained chondrocytes isolated from one another by an extensive extracellular matrix consisting of cartilage type collagen fibrils and proteoglycan granules. The differentiation of the mesenchyme cells to chondrocytes was also characterized by the loss of a 240,000-MW cell surface glycoprotein and the appearance of an 80,000-MW surface protein. The addition of vitamin A to the medium on Day 1 inhibited chondrogenesis. The cells were closely packed together, and the limited extracellular space contained thick, banded collagen fibrils with no proteoglycan granules. The cells exhibited extensive areas of close membrane contact and specialized junctions. Vitamin A-treated cultures also retained the 240,000-MW surface glycoprotein and retarded the appearance of the 80,000-MW cell surface protein. The results of this study suggest that cell surface features normally present on mesenchyme cells are maintained and exaggerated by vitamin A.     

Selenium supplementation of lactating dairy cows: effects on milk production and total selenium content and speciation in blood, milk and cheese

Forty multiparous Holstein cows were used in a 16-week continuous design study to determine the effects of either selenium (Se) source, selenised yeast (SY) (derived from a specific strain of Saccharomyces cerevisiae CNCM I-3060) or sodium selenite (SS), or Se inclusion rate in the form of SY in the diets of lactating dairy cows on the Se concentration and speciation in blood, milk and cheese. Cows received ad libitum a total mixed ration (TMR) with a 1 : 1 forage : concentrate ratio on a dry matter (DM) basis. There were four diets (T1 to T4), which differed only in either source or dose of Se additive. Estimated total dietary Se for T1 (no supplement), T2 (SS), T3 (SY) and T4 (SY) was 0.16, 0.30, 0.30 and 0.45 mg/kg DM, respectively. Blood and milk samples were taken at 28-day intervals and at each time point there were positive linear effects of Se in the form of SY on the Se concentration in blood and milk. At day 112, blood and milk Se values for T1 to T4 were 177, 208, 248 and 279 ± 6.6 and 24, 38, 57 and 72 ± 3.7 ng/g fresh material, respectively, and indicate improved uptake and incorporation of Se from SY. In whole blood, selenocysteine (SeCys) was the main selenised amino acid and the concentration of selenomethionine (SeMet) increased with the increasing inclusion rate of SY. In milk, there were no marked treatment effects on the SeCys content, but Se source had a marked effect on the concentration of SeMet. At day 112, replacing SS (T2) with SY (T3) increased the SeMet concentration of milk from 36 to 111 ng Se/g and its concentration increased further to 157 ng Se/g dried sample as the inclusion rate of SY increased further (T4) to provide 0.45 mg Se/kg TMR. Neither Se source nor inclusion rate affected the keeping quality of milk. At day 112, milk from T1, T2 and T3 was made into a hard cheese and Se source had a marked effect on total Se and the concentration of total Se comprised as either SeMet or SeCys. Replacing SS (T2) with SY (T3) increased total Se, SeMet and SeCys content in cheese from 180 to 340 ng Se/g, 57 to 153 ng Se/g and 52 to 92 ng Se/g dried sample, respectively. The use of SY to produce food products with enhanced Se content as a means of meeting the Se requirements is discussed.     

Diversity,Abundance and Community Structure of Benthic Macro- and Megafauna on the Beaufort Shelf and Slope

Diversity and community patterns of macro- and megafauna were compared on the Canadian Beaufort shelf and slope. Faunal sampling collected 247 taxa from 48 stations with box core and trawl gear over the summers of 2009–2011 between 50 and 1,000 m in depth. Of the 80 macrofaunal and 167 megafaunal taxa, 23% were uniques, present at only one station. Rare taxa were found to increase proportional to total taxa richness and differ between the shelf ( 100 m) where they tended to be sparse and the slope where they were relatively abundant. The macrofauna principally comprised polychaetes with nephtyid polychaetes dominant on the shelf and maldanid polychaetes (up to 92% in relative abundance/station) dominant on the slope. The megafauna principally comprised echinoderms with Ophiocten sp. (up to 90% in relative abundance/station) dominant on the shelf and Ophiopleura sp. dominant on the slope. Macro- and megafauna had divergent patterns of abundance, taxa richness ( diversity) and diversity. A greater degree of macrofaunal than megafaunal variation in abundance, richness and diversity was explained by confounding factors: location (east-west), sampling year and the timing of sampling with respect to sea-ice conditions. Change in megafaunal abundance, richness and diversity was greatest across the depth gradient, with total abundance and richness elevated on the shelf compared to the slope. We conclude that megafaunal slope taxa were differentiated from shelf taxa, as faunal replacement not nestedness appears to be the main driver of megafaunal diversity across the depth gradient.     

Genetic clues to the origin of the apple

Molecular genetic markers complement archaeological, breeding and geographical investigations of the origins, history and domestication of plants. With increasing access to wild apples from Central Asia, along with the use of molecular genetic markers capable of distinguishing between species, and explicit methods of phylogeny reconstruction, it is now possible to test hypotheses about the origin of the domesticated apple. Analyses of nuclear rDNA and chloroplast DNA (cpDNA) sequences indicate that the domesticated apple is most closely related to series Malus species. Moreover, the occurrence of a shared 18-bp duplication in the cpDNAs of wild and cultivated apple supports the close relationship between them. Hypotheses about the hybridization and the origin of the domesticated apple cannot be rejected completely until more variable, phylogenetically informative markers are found.     

Realizing the allosteric potential of the tetrameric protein kinase A RIα holoenzyme

PKA holoenzymes containing two catalytic (C) subunits and a regulatory (R) subunit dimer are activated cooperatively by cAMP. While cooperativity involves the two tandem cAMP binding domains in each R-subunit, additional cooperativity is associated with the tetramer. Of critical importance is the flexible linker in R that contains an inhibitor site (IS). While the IS becomes ordered in the R:C heterodimer, the overall conformation of the tetramer is mediated largely by the N-Linker that connects the?D/D domain to the IS. To understand how the N-Linker contributes to assembly of tetrameric holoenzymes, we engineered a monomeric RIα that contains most of the N-Linker, RIα(73-244), and crystallized a holoenzyme complex. Part of the N-linker is now ordered by interactions with a symmetry-related dimer. This complex of two symmetry-related dimers forms a tetramer that reveals novel mechanisms for allosteric regulation and has many features associated with full-length holoenzyme. A model of the tetrameric holoenzyme, based on this structure, is consistent with previous small angle X-ray and neutron scattering data, and is validated with new SAXS data and with an RIα mutation localized to a novel interface unique to the tetramer.