Membrane Potential and Catecholamine Secretion by Bovine Adrenal Chromaffin Cells: Use of Tetraphenylphosphonium Distribution and Carbocyanine Dye Fluorescence

Changes in plasma membrane potential of isolated bovine adrenal chromaffin cells were measured independently by two chemical probe methods and related to corresponding effects on catecholamine secretion. The lipophilic cation tetraphenylphosphonium (TPP+) and the carbocyanine dye 3,3'-dipropylthiadicarbocyanine [DiS-C3-(5)] were used. The necessity of evaluating the subcellular distribution of TPP+ among cytoplasmic, mitochondrial, secretory granule, and bound compartments was demonstrated and the resting plasma membrane potential determined to be -55 mV. The relationship between membrane potential and catecholamine secretion was determined in response to variations in extracellular K+ and to the presence of several secretagogues including cholinergic receptor ligands, veratridine, and ionophores for Na+ and K+. The dependence of potential on K+ concentration fit the Goldman constant field equation with a Na/K permeability ratio of 0.1. The dependence of both K+- and veratridine-evoked catecholamine secretion on membrane potential exhibited a potential threshold of about -40 mV before a significant rise in secretion occurred. This is likely related to the threshold for opening of voltage-sensitive Ca2+ channels. Acetylcholine and nicotine evoked a large secretory response without a sufficiently sustained depolarization to be detectable by the relatively slow potential sensitive chemical probes. Decamethonium induced a detectable depolarization of the chromaffin cells. Veratridine and gramicidin evoked both membrane depolarization and catecholamine release. By contrast the K ionophore valinomycin evoked significant levels of secretion without any depolarization. This is consistent with its utilization of an intracellular source of Ca2+ and the independence of its measured secretory response on extracellular Ca2+.     

Three dimensional typological studies using scanning electron microscopy for characterization of Termitomyces pellets obtained from submerged growth conditions

There are gaps in existing understanding of fungal pellet growth dynamics. We used scanning electron microscopy (SEM) for morphological characterization of the biomass organization of Termitomyces pellets for seven species: T. microcarpus (TMI1), T. albuminosus (TAL1, TAL2), T. striatus (TSTR), T. aurantiacus (TAUR), T. heimii (THE1, THE2), T. globulus (TGLO) and T. clypeatus (TCL1, TCL2, TCL3, TCL4, TCL5). We assessed the utility of SEM for morphological and structural characterization of Termitomyces spp. in three dimensional (3D) pellet form to identify ideal pellet morphology for industrial use. Typological classification of Termitomyces species was based on furrows, isotropy, total motifs and fractal dimensions. The pellets formed were entangled and exhibited highly compacted mycelial mass with microheterogeneity and microporosity. The mean density of furrows of Termitomyces species was between 10,000 and 11,300 cm/cm², percentage isotropy was 30?80 and total motifs varied from 300 to 2500. TGLO exhibited the highest furrow mean density, 11243 cm/cm², which indicated a compact, cerebroid structure with complex ridges and furrows, whereas TAL2 exhibited the lowest furrow density. TMI1a exhibited a high percentage isotropic value, 74.6, TSTR exhibited the lowest, 30.9. Total motif number also was used as a typological classification parameter. Fractal values were 2.64?2.78 for various submerged conditions of Termitomyces species. TAL1 exhibited the highest fractal dimension and TAL2 the lowest, which indicates the complexity of branching patterns. Three-dimensional SEM image analysis can provide insight into pellet micromorphology and is a powerful tool for exploring topographical details of pellets.     

Time, gender, and seasonality in vervet activity: A chronobiological approach

A chronobiological approach has been used to highlight the issue of how climatic factors and breeding seasonality may affect male and female diurnal activity budget. We investigated gender differences for vervet monkeys in the diurnal distribution of feeding, locomotion, inactivity, and social grooming within and between birth, pre-mating, and mating seasons. The main climatic traits were that days were shorter, drier, and cooler in the mating season. Non-parametric statistics showed that female activities changed with time of day to a greater extent than did male activities. When the constraints imposed by climatic factors and mate competition increased, from the birth to the mating season, male maintenance activities were more independent of the time of day whereas females continued to vary. Gender differences in timing were therefore greater in the mating season, except for social grooming; males and females were more coordinated in diurnal timing of their grooming in the mating season. It is argued that these changes may result in reduced levels of male-female contest competition, and may be related to higher levels of male-female socialization in the mating season. Finally, it is inferred that an out-of-phase synchronization between inactivity and feeding for males in the birth season, and for females in the mating season, may result from each sex investing more time feeding at the expense of resting in those periods.     

Sea urchin Hox genes: insights into the ancestral Hox cluster

We describe the Hox cluster in the radially symmetric sea urchin and compare our findings to what is known from clusters in bilaterally symmetric animals. Several Hox genes from the direct-developing sea urchin Heliocidaris erythrogramma are described. CHEF gel analysis shows that the Hox genes are clustered on a < or = 300 kilobase (kb) fragment of DNA, and only a single cluster is present, as in lower chordates and other nonvertebrate metazoans. Phylogenetic analyses of sea urchin, amphioxus, Drosophila, and selected vertebrate Hox genes confirm that the H. erythrogramma genes, and others previously cloned from other sea urchins, belong to anterior, central, and posterior groups. Despite their radial body plan and lack of cephalization, echinoderms retain at least one of the anterior group Hox genes, an orthologue of Hox3. The structure of the echinoderm Hox cluster suggests that the ancestral deuterostome had a Hox cluster more similar to the current chordate cluster than was expected Sea urchins have at least three Abd-B type genes, suggesting that Abd-B expansion began before the radiation of deuterostomes.      

A microscopy study of hyphal growth of Penicillium rubens on gypsum under dynamic humidity conditions

To remediate indoor fungal growth, understanding the moisture relations of common indoor fungi is crucial. Indoor moisture conditions are commonly quantified by the relative humidity (RH). RH is a major determinant of the availability of water in porous indoor surfaces that fungi grow on. The influence of steady‐state RH on growth is well understood. Typically, however, the indoor RH constantly changes so that fungi have to endure frequent periods of alternating low and high RH. Knowledge of how common indoor fungi survive and are affected by the low‐RH periods is limited. In particular, the specific effects of a drop in RH on the growth of the mycelium remain unclear. In this work, video microscopy was used to monitor hyphal growth of Penicillium rubens on gypsum substrates under controlled dynamic humidity conditions. The effect of a single period of low RH (RH = 50–90%) interrupting favourable conditions (RH = 97%) was tested. It was found that hyphal tips ceased to extend when exposed to any tested decrease in RH. However, new hyphal growth always emerges, seemingly from the old mycelium, suggesting that this indoor fungus does not rely only on conidia to survive the humidity patterns considered. These findings are a fundamental step in unravelling the effect of RH on indoor fungal growth.     

Amino acid metabolism of bovine blastocysts: a biomarker of sex and viability

The ratio of male/female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling, a noninvasive marker of developmental potential to compare the effect of sex on the metabolism of bovine blastocysts conceived in vivo and in vitro. Blastocysts were incubated individually for 24 hr in a close‐to‐physiological mixture of amino acids and the depletion or appearance of 18 amino acids measured using HPLC. Blastocysts were then sexed by PCR. Amino acid depletion by in vitro‐produced blastocysts and expanded blastocysts was higher than in embryos conceived in vivo (P = 0.02). When cultured in vitro, female embryos exhibited increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro‐produced blastocysts exhibited sex‐specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo‐produced, in 2 out of 18. These differences had disappeared by the expanded blastocyst stages. We have also shown that amino acid metabolism can predict the ability of bovine zygotes to develop to the blastocyst stage, providing “proof of principle” for the use of this technology in clinical IVF to select single embryos for transfer and thereby avoid the problem of multiple births. Mol. Reprod. Dev. 77: 285–296, 2010. © 2010 Wiley‐Liss, Inc.     

Reduced EGFR signaling in progenitor cells of the adult subventricular zone attenuates oligodendrogenesis after demyelination

Neural progenitor cells that express the NG2 proteoglycan are present in different regions of the adult mammalian brain where they display distinct morphologies and proliferative rates. In the developing postnatal and adult mouse, NG2(+) cells represent a major cell population of the subventricular zone (SVZ). NG2(+) cells divide in the anterior and lateral region of the SVZ, and are stimulated to proliferate and migrate out of the SVZ by focal demyelination of the corpus callosum (CC). Many NG2(+) cells are labeled by GFP-retrovirus injection into the adult SVZ, demonstrating that NG2(+) cells actively proliferate under physiological conditions and after demyelination. Under normal physiological conditions and after focal demyelination, proliferation of NG2(+) cells is significantly attenuated in wa2 mice, which are characterized by reduced signaling of the epidermal growth factor receptor (EGFR). This results in reduced SVZ-to-lesion migration of NG2(+) cells and oligodendrogenesis in the lesion. Expression of vascular endothelial growth factor (VEGF) and EGFR ligands, such as heparin binding-EGF and transforming growth factor alpha, is upregulated in the SVZ after focal demyelination of the CC. EGF-induced oligodendrogenesis and myelin protein expression in wild-type SVZ cells in culture are significantly attenuated in wa2 SVZ cells. Our results demonstrate that the response of NG2(+) cells in the SVZ and their subsequent differentiation in CC after focal demyelination depend on EGFR signaling.     

Sustained NPY Overexpression in the PVN Results in Obesity via Temporarily Increasing Food Intake

Increasing neuropeptide Y (NPY) signaling in the paraventricular nucleus (PVN) by recombinant adeno‐associated virus (rAAV)‐mediated overexpression of NPY in rats, results in hyperphagia and obesity in rats. To determine the importance of hyperphagia in the observed obesity phenotype, we pair‐fed a group of AAV‐NPY‐injected rats to AAV‐control‐injected rats and compared parameters of energy balance to ad libitum fed AAV‐NPY‐injected rats. For 3 weeks, AAV‐NPY‐injected rats, received the same amount of food as ad libitum‐fed rats injected with control rAAV They did not gain more body weight than these controls. When allowed access to food ad libitum, these AAV‐NPY‐injected rats increased food intake, which subsequently decreased when rats reached the same body weight as AAV‐NPY‐injected rats that were fed ad libitum for the entire study. These data indicate that overexpression of NPY in the PVN results in obesity by increasing food intake until a certain body weight is achieved.     

Occasional intraguild predation structuring small mammal assemblages: the marsupial Didelphis aurita in the Atlantic Forest of Brazil

The didelphid marsupial, Didelphis aurita, is suggested as an intraguild predator and as key‐species in small mammal assemblages of the Atlantic Forest of Brazil. The field experiments required to test this hypothesis are complex to implement, but the recent revival of regression methods offers a viable alternative. Here we use the dynamic and static regression methods to determine the importance of D. aurita as a competitor and intraguild predator. Capture–recapture data from two localities in the Rio de Janeiro State were used, Garrafão (municipality of Guapimirim), a coastal forest of the Serra do Mar, and Barra de Maricá, a costal sand dune vegetation. Population and microhabitat variables were monitored from April 1997 to April 2003 in Garrafão, and from January 1986 to July 1990 in Barra de Maricá. Microhabitat variables were related to Canopy, Plant, Litter and Rock covers, Obstruction from 0 to 1.5 m, and Number of logs. Exploitation competition was tested by the dynamic method, which models the effects of D. aurita on the per capita growth rate of a species. Interference by predation or competition was tested by the static method, where the abundance of D. aurita at trap stations was regressed against the abundance of other small mammals, after removal of any variation associated with microhabitat factors. Exploitation competition was not detected, but the interference of D. aurita was pervasive, affecting all small mammals studied in the two localities. The clear avoidance of D. aurita by all small mammals tested in two localities of different physiognomies indicates that it functions as an intraguild predator, even if actual predation by D. aurita is an occasional event.