Glycosaminoglycan origin and structure revealed by multivariate analysis of NMR and CD spectra

Principal component analysis (PCA) is a method of simplifying complex datasets to generate a lower number of parameters, while retaining the essential differences and allowing objective comparison of large numbers of datasets. Glycosaminoglycans (GAGs) are a class of linear sulfated carbohydrates with diverse sequences and consequent complex conformation and structure. Here, PCA is applied to three problems in GAG research: (i) distinguishing origins of heparin preparations, (ii) structural analysis of heparin derivatives, and (iii) classification of chondroitin sulfates (CS). The results revealed the following. (i) PCA of heparin (13)C NMR spectra allowed their origins to be distinguished and structural differences were identified. (ii) Analysis of the information-rich (1)H and (13)C NMR spectra of a series of systematically modified heparin derivatives uncovered underlying properties. These included the presence of interactions between residues, providing evidence that a degree of degeneracy exists in linkage geometry and that a different degree of variability exists for the two types of glycosidic linkage. The relative sensitivity of each position (C or H nucleus) in the disaccharide repeating unit to changes in O-, N-sulfation and N-acetylation was also revealed. (iii) Analysis of the (1)H NMR and CD spectra of a series of CS samples from different origins allowed their structural classification and highlighted the power of employing complementary spectroscopic methods in concert with PCA.     

Bacteria beneath the West Antarctic Ice Sheet

Subglacial environments, particularly those that lie beneath polar ice sheets, are beginning to be recognized as an important part of Earth's biosphere. However, except for indirect indications of microbial assemblages in subglacial Lake Vostok, Antarctica, no sub-ice sheet environments have been shown to support microbial ecosystems. Here we report 16S rRNA gene and isolate diversity in sediments collected from beneath the Kamb Ice Stream, West Antarctic Ice Sheet and stored for 15 months at 4°C. This is the first report of microbes in samples from the sediment environment beneath the Antarctic Ice Sheet. The cells were abundant (∼10⁷ cells g⁻¹) but displayed low diversity (only five phylotypes), likely as a result of enrichment during storage. Isolates were cold tolerant and the 16S rRNA gene diversity was a simplified version of that found in subglacial alpine and Arctic sediments and water. Although in situ cell abundance and the extent of wet sediments beneath the Antarctic ice sheet can only be roughly extrapolated on the basis of this sample, it is clear that the subglacial ecosystem contains a significant and previously unrecognized pool of microbial cells and associated organic carbon that could potentially have significant implications for global geochemical processes.     

Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection

One of the first steps in characterizing heparan sulfate (HS) and its close relative heparin is to conduct disaccharide composition analysis. This provides an overall picture of the structure of the polysaccharide in terms of its constituent disaccharides. This is of importance, for example, in the initial characterization of spatially and temporally regulated structures. Two protocols for conducting disaccharide analysis are presented here, both exploiting exhaustive digestion of the polysaccharide, yielding constituent disaccharides, by bacterial heparin lyases. The first method, suitable for microgram quantities of material, relies on the separation of the disaccharides by high-performance liquid chromatography (HPLC) coupled to ultraviolet absorbance detection and can be performed in 2 d. The second exploits reducing end-labeling with the fluorophore BODIPY hydrazide, separation by HPLC, and subsequent fluorescence detection and quantitation. The latter is a high-sensitivity method that requires nanograms of starting material and has a detection limit in the low fmol range, and is thus the most sensitive method for disaccharide compositional analysis of HS yet reported. Fluorescence detection can be routinely carried out in 3 d.     

In vitro culture of shoots of Pinus caribaea on a liquid medium

Shoot apices of a clone of Pinus caribaea Morelet were cultured and multiplied in vitro by supporting them with their basal cut ends immersed in a liquid nutrient medium.The initial heights of explants and their initial numbers of leaves were positively correlated with the numbers of buds and shoots produced by the explants after a bud induction phase and after a shoot elongation phase. The final numbers of buds and shoots were positively correlated with reductions in the quantities of phosphorus detected in the media and negatively correlated with the numbers of brown leaves produced on the explants.In a comparison between the growth of shoot explants on liquid and solid media, shoots incubated on the liquid medium showed significantly greater increases in length in a four-week period than those cultured on solid medium.This technique, using liquid media, provides a system in which both the nutrient utilization and the growth rates of isolated pine tissues can be readily assessed. Furthermore, the multiplication rate of the tissue can be predicted following the observation of correlated characters early in the micropropagation cycle.     

Binding of antigen-specific, H-2-restricted T cell hybridomas to antigen-pulsed adherent cell monolayers

Two methods were investigated for studying the binding of radiolabeled hybridoma T cells to antigen (Ag) and H-2 products for which they bore receptors. In both cases hybridoma T cells were labeled with tritiated thymidine. In one method labeled cells were added to adherent splenic cells prepulsed with antigen, and the mixture was incubated overnight at 37 degrees C before nonadherent cells were gently washed away. The percent of adherent hybridoma T cells was then estimated by harvesting the adherent monolayers and measuring tritium counts bound. In a second method radiolabeled hybridoma T cells were added to adherent antigen-pulsed B cell lymphomas or hybridomas for between 15 min and 1 hr at 37 degrees C before removal of nonadherent cells and harvesting of the adherent monolayers. In both cases binding was both antigen- and I-region specific. In the second case binding was also rapid; significant binding could be measured after 15 min incubation. These techniques were used to study subclones of one of our T cell hybridomas that were thought by a functional assay (interleukin 2 release) to have lost receptors for Ag/H-2. It was found that subclones of the hybridoma that no longer secreted interleukin 2 in response to Ag/H-2, even though they continued to secrete interleukin 2 in response to concanavalin A, also no longer bound specifically to Ag-pulsed monolayers of the appropriate H-2 type. This confirmed the idea that these subclones had lost the ability to synthesize receptors for Ag/H-2. It is hoped that assays of this type will be useful in the future for the study of Ag/H-2 receptors on T cells.     

Crystallization of the A alpha subunit of protein phosphatase 2A.

The A alpha subunit of human protein phosphatase 2A forms crystals in space group P2(1) with cell dimensions a = 104.0, b = 174.9, c = 168.2 A, and beta angle = 90.2 degrees. At cryogenic temperatures, the crystals diffracted to a resolution limit of approximately 3.0 A. Based on the unit cell dimensions and a calculated molecular mass of 65,277 Da, the Matthews coefficient suggests eight molecules per asymmetric unit. Two native data sets were collected to a nominal resolution of 3.0 A and merged to provide a set that is 93% complete, with Rsym of 9.9%.     

Mass transfer phenomena in an airlift reactor: effects of solids loading and temperature

Gas holdups and volumetric mass transfer coefficients were measured in a concentric tube airlift reactor designed for the microbial desulfurization of coal. The solutions studied were comprised of an acidified basal salts solution containing thirteen different weight percentages (0 to 40) of coal (74 mum Ohio #1) at three different temperatures (30, 50, and 72 degrees C). Gas holdup epsilon(G) decreased with solids loading for the entire range studied. An enhancement in the volumetric mass transfer coefficient K(L)a with respect to that in pure solution was observed from zero to approximately 5 wt % (solids volume fraction epsilon(s) = 0.035), the maximum enhancement occurring at approximately 2 wt % (epsilon(s) = 0.014). At higher solids fractions, the mass transfer coefficient decreased with further solids additions. Gas holdups and the mass transfer coefficients increased with temperature over the studied range. The K(L)a and epsilon(G) were correlated to three process variables separately and the separate correlations combined to yield generalized correlations for the mass transfer coefficient and gas holdup for this system. The correlations may be used for design, operation, and ost estimation of such systems.     

Control of luteolysis in the one-humped camel (Camelus dromedarius)

Blood plasma concentrations of 13,14-dihydro-15-keto PGF2 alpha (PGFM) were measured in groups of mature non-pregnant and pregnant camels to study PGF2 alpha release patterns around the time of luteolysis and the timing of the signal for pregnancy recognition. Injection of each of four camels with 10 and 50 mg of PGF2 alpha showed clearly that five times the dose of exogenous hormone produced five times the amount of PGFM in peripheral plasma, thereby indicating that, as in other animal species, PGFM is the principal metabolite of PGF2 alpha in the camel. Serial sampling of three non-pregnant camels on each of days 8, 10 and 12, and three pregnant camels on day 10, after ovulation for 8 h showed a significant (P < 0.05) rise in mean plasma PGFM concentrations only on day 10 in the non-pregnant, but not the pregnant, animals. A single intravenous injection of 20, 50 or 100 iu oxytocin given to three groups of three non-pregnant camels on day 10 after ovulation did not increase their basal serum PGFM concentrations. However, daily treatment of six non-pregnant camels between days 6 and 15 (n = 3) or 20 (n = 3) after ovulation with 1-2 g of the prostaglandin synthetase inhibitor, meclofenamic acid, inhibited PGF2 alpha release and thereby resulted in continued progesterone secretion throughout the period of meclofenamic acid administration. These results showed that, as in other large domestic animal species, release of PGF2 alpha from, presumably, the endometrium controls luteolysis in the dromedary camel. Furthermore, reduction in the amount of PGF2 alpha released is associated with luteal maintenance and the embryonic signal for maternal recognition of pregnancy must be transmitted before day 10 after ovulation if luteostasis is to be achieved. However, the results also indicate that, in contrast to ruminants, the release of endometrial PGF2 alpha in the non-pregnant camel may not be controlled by the release of oxytocin.     

Body size and abundance relationship: an index of diversity for herbivores

It is evident to any biologist that small-bodied species within a given higher taxon (order, class, phylum, etc.) tend to be represented by more individuals. Hence small-bodied species are generally more abundant than large-bodied species. We analyzed large herbivore species data collected in Kenyan rangelands. An index of biological diversity derived from the negative relation between animal species body size and its local abundance is proposed. We compared the new index with species abundances at landscape scale (10 × 10 km) in individual districts, as well as in the combined regional data. The results show a consistently strong positive relation between the new diversity index and species abundances. The proposed diversity index has the advantage of incorporating information on species abundances without the need for time-consuming surveys.     

Fine-scale spatial distribution of plants and resources on a sandy soil in the Sahel

Three species, wheat, maize and cotton, were grown in pots and subjected to high (85–100% field capacity, _F), medium (65–85% _F) and low (45–65% _F) soil moisture treatments and high (700 l l^–1) and low (350 l l^–1) CO₂ concentrations. Biomass production, photosynthesis, evapotranspiration and crop water use efficiency were investigated. Results showed that the daily photosynthesis rate was increased more in wheat and cotton at high [CO₂] than in maize. In addition, differences were more substantial at low soil water treatment than at high soil water treatment. The daily leaf transpiration was reduced significantly in the three crops at the high CO₂ concentration. The decrease at low soil water was smaller than at high soil water. Crop biomass production responses showed a pattern similar to photosynthesis, but the CO₂-induced increase was more pronounced in root production than shoot production under all soil water treatments. Low soil water treatment led to more root biomass under high [CO₂] than high soil water treatment. CO₂ enrichment caused a higher leaf water use efficiency (WUE) of three crops and the increase was more significant in low than in high soil water treatment. Crop community WUE was also increased by CO₂ enrichment, but the increase in wheat and cotton was much greater than in maize. We conclude that at least in the short-term, C₃ plants such as wheat and cotton may benefit from CO₂ enrichment especially under water shortage condition.