Cell patterning in branched and unbranched fruiting bodies of the cellular slime mold Polysphondylium pallidum

Cell patterning, the percentage of spores and stalk cells, was measured in branched and unbranched asexual fruiting bodies of Polysphondylium pallidum. Unlike D. discoideum, where small and large fruiting bodies are more stalky than average-sized fruiting bodies, the overall cell patterning was the same in branched and unbranched fruiting bodies of all sizes in P. pallidum. Light greatly increased the numbers of fruiting bodies in P. pallidum per unit area (or decreased aggregation territory size) so that most fruiting bodies formed in the light were small and unbranched. By contrast, light had little effect on the cell patterning of P. pallidum, although there was a slight increase in the percentage of stalk cells in the light compared to the dark. This indicates that the mechanisms governing light sensitivity of aggregation territory size and cell patterning have different components in P. pallidum. The accuracy of cell patterning of individual branches of branched fruiting bodies was so imprecise as to leave doubt that patterning is occurring at the branch level. Individual whorls of branched fruiting bodies had a greater percentage spores (90%) than whole fruiting bodies (78%) and the cell patterning was relatively imprecise. Only in whole fruiting bodies was the spore:stalk ratio highly correlated. These findings are consistent with cell pattern determination operating at the whole aggregate level, rather than at the individual whorl or branch level in P. pallidum.     

Characterisation of Rubus niveus: a prerequisite to its biological control in oceanic islands

This study is intended to inform the search for a biocontrol solution to stem the spread of Rubus niveus, a morphologically-diverse and invasive Rubus species which has become an aggressive invader in areas of the world outside its native range. The fragile biodiversity of islands can be especially threatened by this plant – exemplars of this are the Galápagos archipelago and Hawaii. This report focuses on the morphological and molecular work carried out to establish the homogeneity or intra-specific variation of R. niveus populations within the Galápagos Islands, as well as their kinship with plants of the same name found in Asia. The premise of this study is that genetic closeness between these populations offers increased probability that pathogens of R. niveus in its native range will also provide effective control in the target environment. Increased knowledge of the genetic make-up of R. niveus in both its native and naturalised locations will also help to assess the feasibility of using organisms which have been found to be effective against other Rubus species, but not hitherto considered for R. niveus itself.     

Distribution of active protein kinase C in smooth muscle.

To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 microM carbachol for 1 min, 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 micrometer 4-alpha phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-alpha PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction.     

Human metabolic phenotypes link directly to specific dietary preferences in healthy individuals

Individual human health is determined by a complex interplay between genes, environment, diet, lifestyle, and symbiotic gut microbial activity. Here, we demonstrate a new "nutrimetabonomic" approach in which spectroscopically generated metabolic phenotypes are correlated with behavioral/psychological dietary preference, namely, "chocolate desiring" or "chocolate indifferent". Urinary and plasma metabolic phenotypes are characterized by differential metabolic biomarkers, measured using 1H NMR spectroscopy, including the postprandial lipoprotein profile and gut microbial co-metabolism. These data suggest that specific dietary preferences can influence basal metabolic state and gut microbiome activity that in turn may have long-term health consequences to the host. Nutrimetabonomics appears as a promising approach for the classification of dietary responses in populations and personalized nutritional management.     

Nutritional metabonomics: applications and perspectives

Nowadays, nutrition focuses on improving health of individuals through diet. Current nutritional research aims at health promotion, disease prevention, and performance improvement. Modern analytical platforms allow the simultaneous measurement of multiple metabolites providing new insights in the understanding of the functionalities of cells and whole organisms. Metabonomics, "the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modifications", provides a systems approach to understanding global metabolic regulations of organisms. This concept has arisen from various applications of NMR and MS spectroscopies to study the multicomponent metabolic composition of biological fluids, cells, and tissues. The generated metabolic profiles are processed by multivariate statistics to maximize the recovery of information to be correlated with well-determined stimuli such as dietary intervention or with any phenotypic data or diet habits. Metabonomics is thus uniquely suited to assess metabolic responses to deficiencies or excesses of nutrients and bioactive components. Furthermore, metabonomics is used to characterize the metabolic phenotype of individuals integrating genetic polymorphism, metabolic interactions with commensal and symbiotic partners such as gut microflora, as well as environmental and behavioral factors including dietary preferences. This paper reports several experimental key aspects in nutritional metabonomics, reviews its applications employing targeted and holistic approach analysis for the study of the metabolic responses following dietary interventions. It also reports the assessment of intra- and inter-individual variability in animal and human populations. The potentialities of nutritional metabonomics for the discovery of new biomarkers and the characterization of metabolic phenotypes are discussed in a context of their possible utilizations for personalized nutrition to provide health maintenance at the individual level.     

Rapid Ornithine Decarboxylase Test for the Identification of Enterobacteriaceae

Conventional methods for detecting ornithine decarboxylase activity require an extended period of incubation. However, with a few simple modifications, accurate results were obtained within a few hours rather than several days. The broth medium was modified, primarily by omitting glucose and by decreasing the pH to 5.5. A 1-ml amount of this broth was inoculated with one colony and then overlaid with sterile mineral oil. Within 2 to 4 hr, the pH increased if ornithine was decarboxylated, thus changing the color of the internal pH indicator to a dark purple. If the amino acid was not decarboxylated, the pH decreased to pH 5.0 to 5.2, enough to give a definite yellow color. With 347 selected clinical isolates, the rapid test gave results identical to those obtained in 1 to 4 days with Moeller's decarboxylase medium. Less reliable results were obtained with Difco's decarboxylase medium with 0.3% agar which was stabinoculated and read after 18 to 24 hr without a mineral oil seal. The rapid ornithine decarboxylase test represents a simple, accurate technique which is well suited for the clinical microbiology laboratory.     

Replacing the Factor VIII C1 Domain with a Second C2 Domain Reduces Factor VIII Stability and Affinity for Factor IXa

Factor VIII (FVIII) consists of a heavy chain (A1(a1)A2(a2)B domains) and light chain ((a3)A3C1C2 domains). To gain insights into a role of the FVIII C domains, we eliminated the C1 domain by replacing it with the homologous C2 domain. FVIII stability of the mutant (FVIII_C2C2) as measured by thermal decay at 55 °C of FVIII activity was markedly reduced (∼11-fold), whereas the decay rate of FVIIIa due to A2 subunit dissociation was similar to WT FVIIIa. The binding affinity of FVIII_C2C2 for phospholipid membranes as measured by fluorescence resonance energy transfer was modestly lower (∼2.8-fold) than that for WT FVIII. Among several anti-FVIII antibodies tested (anti-C1 (GMA8011), anti-C2 (ESH4 and ESH8), and anti-A3 (2D2) antibody), only ESH4 inhibited membrane binding of both WT FVIII and FVIII_C2C2. FVIIIa cofactor activity measured in the presence of each of the above antibodies was examined by FXa generation assays. The activity of WT FVIIIa was inhibited by both GMA8011 and ESH4, whereas the activity of FVIII_C2C2 was inhibited by both the anti-C2 antibodies, ESH4 and ESH8. Interestingly, factor IXa (FIXa) binding affinity for WT FVIIIa was significantly reduced in the presence of GMA8011 (∼10-fold), whereas the anti-C2 antibodies reduced FIXa binding affinity of FVIII_C2C2 variant (∼4-fold). Together, the reduced stability plus impaired FIXa interaction of FVIII_C2C2 suggest that the C1 domain resides in close proximity to FIXa in the FXase complex and contributes a critical role to FVIII structure and function.