Partly ordered synthesis and degradation of glycogen in cultured rat myotubes.

The following questions concerning glycogen synthesis and degradation were examined in cultured rat myotubes. 1) Is synthesis and degradation of the individual glycogen molecule a strictly ordered process, with the last glucosyl unit incorporated into the molecule being the first to be released (the last-in-first-out principle), or is it a random process? 2) Are all glycogen molecules in skeletal muscle synthesized and degraded in phase (simultaneous order) or out of phase (sequential order)? Basal glycogen stores were minimized by fasting and were subsequently replenished in two intervals, the first (0-0.5 h) with tritium-labeled and the second (0.5-3 h) with carbon-labeled glucose as precursor. Glycogen degradation was initiated by addition of forskolin. The kinetics of glycogen accumulation as well as degradation could be approximated by monoexponential equations with rate constants of 0.81 and 1.39 h(-1), respectively. The degradation of glycogen largely followed the last-in-first-out principle, particularly in the initial period. Analysis of the size of the glycogen molecules and the beta-dextrin limit during glycogen accumulation and degradation showed that both synthesis and degradation of glycogen molecules are largely sequential and the small deviation from this order is most pronounced at the beginning of the accumulation and at the end of the degradation period. This pattern may reflect the number of synthase and phosphorylase molecules and fits well with the role of glycogen in skeletal muscle as a readily available energy store and with the known structure of the glycogen molecule. It is emphasized that the observed nonlinear relation between the change in glycogen concentration and release of label during glycogen degradation may have important practical consequences for interpretation of experimental data.     

Effects of planted tree fallows on soil nitrogen dynamics,above-ground and root biomass,N2-fixation and subsequent maize crop productivity in Kenya

The effects of planted fallows of Sesbania sesban (L.) Merr. and Calliandra calothyrsus (Meissner) on soil inorganic nitrogen dynamics and two subsequent maize crops were evaluated under field conditions in the highlands of eastern Kenya. Continuous unfertilised maize, maize/bean rotation and natural regrowth of vegetation (weed fallow) were used as control treatments. The proportion of symbiotic N₂-fixation was estimated by measuring both leaf ¹⁵N enrichment and whole-plant ¹⁵N enrichment by the ¹⁵N dilution technique for Sesbania and Calliandra, using Eucalyptus saligna (Sm.) and Grevillea robusta (A. Cunn) as reference species. Above- and below-ground biomass and N contents were examined in Sesbania, Calliandra, Eucalyptus and Grevillea 22 months after planting. Both the content of inorganic N in the topsoil and the quantity of N mineralised during rainy seasons were higher after the Sesbania fallows than after the other treatments. Compared to the continuous unfertilised maize treatment, both residual crop yields were significantly higher when mineral N (one application of 60 kg N ha^–1) was added. Furthermore, the second crop following the Sesbania fallow was significantly higher than the continuous maize crop. The above-ground biomass of the trees at final harvest were 31.5, 24.5, 32.5 and 43.5 Mg ha^–1 for the Sesbania, Calliandra, Grevillea and Eucalyptus, respectively. For the total below-ground biomass the values for these same tree species were 11.1, 15.5, 17.7, and 19.1 Mg ha^–1, respectively, of which coarse roots (>2 mm), including tap roots, amounted to 70–90%. About 70–90% of the N in Sesbania, and 50–70% in Calliandra, was derived from N₂-fixation. Estimates based on leaf ¹⁵N enrichment and whole-plant ¹⁵N enrichment were strongly correlated. The N added by N₂-fixation amounted to 280–360 kg N ha^–1 for Sesbania and 120–170 kg N ha^–1 for Calliandra, resulting in a positive N balance after two maize cropping seasons of 170–250 kg N ha^–1 and 90–140 kg N ha^–1, for Sesbania and Calliandra, respectively. All the other treatments gave negative N balances after two cropping seasons. We conclude that Sesbania sesban is a tree species well suited for short duration fallows due to its fast growth, high nutrient content, high litter quality and its ability to fix large amounts of N₂ from the atmosphere.     

Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry

Summary Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG—gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase—isomaltase. The labelling efficiency of RPGG was compared to that of protein A—gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12nm particles, 10.3 and 6.2 particles/µm of length of microvillar membrane, 3.5 and 1.0 particles/µm² of Golgi profile and 5.9 and 2.0 particles/µm² of multivesicular body profile; and for the 6nm particles, 49.6 and 15.7 particles/µm of length of microvillar membrane, 24.4 and 5.0 particles/µm² of Golgi profile and 25.4 and 3.4. particles/µm² of multivesicular body profile. Controls showed very little non-specific gold labelling (<0.02 gold particles/µm² of section).     

Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133⁺ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.     

Evoked Emotions Predict Food Choice

In the current study we show that non-verbal food-evoked emotion scores significantly improve food choice prediction over merely liking scores. Previous research has shown that liking measures correlate with choice. However, liking is no strong predictor for food choice in real life environments. Therefore, the focus within recent studies shifted towards using emotion-profiling methods that successfully can discriminate between products that are equally liked. However, it is unclear how well scores from emotion-profiling methods predict actual food choice and/or consumption. To test this, we proposed to decompose emotion scores into valence and arousal scores using Principal Component Analysis (PCA) and apply Multinomial Logit Models (MLM) to estimate food choice using liking, valence, and arousal as possible predictors. For this analysis, we used an existing data set comprised of liking and food-evoked emotions scores from 123 participants, who rated 7 unlabeled breakfast drinks. Liking scores were measured using a 100-mm visual analogue scale, while food-evoked emotions were measured using 2 existing emotion-profiling methods: a verbal and a non-verbal method (EsSense Profile and PrEmo, respectively). After 7 days, participants were asked to choose 1 breakfast drink from the experiment to consume during breakfast in a simulated restaurant environment. Cross validation showed that we were able to correctly predict individualized food choice (1 out of 7 products) for over 50% of the participants. This number increased to nearly 80% when looking at the top 2 candidates. Model comparisons showed that evoked emotions better predict food choice than perceived liking alone. However, the strongest predictive strength was achieved by the combination of evoked emotions and liking. Furthermore we showed that non-verbal food-evoked emotion scores more accurately predict food choice than verbal food-evoked emotions scores.     

ULTRASTRUCTURE OF GYMNODINIUM AUREOLUM (DINOPHYCEAE): TOWARD A FURTHER REDEFINITION OF GYMNODINIUM SENSU STRICTO

Examination of the ultrastucture of the unarmored dinoflagellate Gymnodinium aureolum (Hulburt) G. Hansen (syn: Gyrodinium aureolum Hulburt) revealed the presence of nuclear chambers, which are specialized differentiations of the nuclear envelope, similar to those described in the type species of Gymnodinium , G. fuscum (Ehrenberg) Stein and certain other Gymnodinium species. The nuclear pores were restricted to these chambers. In the flagellar apparatus a nuclear fibrous connective linked the longitudinal microtubular root and the nucleus. This structure had so far been observed only in Gymnodinium spp. and in the heterotrophic species Actiniscus pentasterias (Ehrenberg) Ehrenberg, Nematodinium armatum (Dogiel) Kofoid et Swezy and Polykrikos kofoidii Chatton. Another unusual feature of G. aureolum was the presence of a striated fiber in the longitudinal flagellum, a feature previously only found in Ceratium furca (Ehrenberg) Claparède et Lachmann and C. tripos (O.F. Müller) Nitzsch. Gymnodinium aureolum also possessed a prominent ventral protrusion associated with the peduncle and containing electron opaque material. It is concluded that G. aureolum belongs to the Gymnodinium sensu stricto group. This may be a temporary classification, however, because G. aureolum and its allies differ from the type species G. fuscum by the presence of a transverse striated root, striated collars, trichocysts, and a peduncle.