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+.     

Effects of Increased Extracellular K on the Elemental Composition and Water Content of Neuron and Glial Cells in Leech CNS

Elemental (Na, Cl, K) and water contents of leech (Macrobdella decora) neurons and glial cells were determined under steady-state exposure to 4, 10, and 20 mM KCl concentrations (bathing media) using x-ray microanalysis for quantitative digital imaging of frozen hydrated and dried cryosections. Effects of furosemide, 5-hydroxytryptamine (5-HT), and ouabain on elemental distribution changes, induced by exposure to 20 mM K, were also determined. Results demonstrated that packet glial cells and neurons accumulated substantial amounts of K that appeared evenly distributed throughout the cytoplasm. Cell water content also increased as a function of increased cytoplasmic K so that the net effect was an unchanged wet-weight K concentration (expressed as millimoles per kilogram wet weight). Dry-weight Na and Cl concentration (expressed as millimoles per kilogram dry weight) increased slightly in glial cells; however, because cell water increased, both Na and Cl (wet-weight) concentrations decreased. Neurons, in contrast, had no significant change in either Na or K on a wet-weight basis, so a relatively constant Na/K ratio was maintained despite a small, but significant, increase in K (dry weight) and cell water. These increases, like those in packet glia, were a function of exposure to different concentrations of extracellular space K. These changes were completely abolished by 10(-4) M ouabain. Neither furosemide nor 5-HT appeared to affect neuronal or glial K wet-weight concentrations. These data show that both glial cells and neurons can act as substantial reservoirs for K while maintaining stable K concentrations (by altering cell water content and elemental composition). This process appears to depend on a functioning Na+, K+-ATPase system.     

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.     

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.      

Allergy to penicillin: fable or fact?

OBJECTIVE--To assess whether, on the basis of one blood test, penicillin allergy might be excluded sufficiently for general practitioners to give oral penicillin to patients claiming a history of penicillin allergy. DESIGN--Prospective study of patients referred by general practitioners. SETTING--Outpatient allergy clinic in a district general hospital. PATIENTS--175 referred patients who gave a history of immediate type reaction to penicillin, of whom 144 attended as requested and 132 completed the investigations. MAIN OUTCOME MEASURES--History and examination, serum radioallergosorbent test to phenoxymethylpenicillin and benzylpenicillin, and oral challenge with penicillin. RESULTS--Of 132 patients, four were confirmed to have penicillin allergy by the radioallergosorbent test and 128 had an oral penicillin challenge without ill effect. CONCLUSIONS--Most patients who gave a history of penicillin allergy are not so allergic, and their actual allergic state should be substantiated whenever feasible. For patients reporting minor or vague reactions negative findings with a radioallergosorbent test to phenoxymethylpenicillin and benzylpenicillin provide sufficient evidence to give oral penicillin safely.     

Phenotypic characterization of Bbs4 null mice reveals age-dependent penetrance and variable expressivity

Bardet-Biedl syndrome (BBS) is a rare oligogenic disorder exhibiting both clinical and genetic heterogeneity. Although the BBS phenotype is variable both between and within families, the syndrome is characterized by the hallmarks of developmental and learning difficulties, post-axial polydactylia, obesity, hypogenitalism, renal abnormalities, retinal dystrophy, and several less frequently observed features. Eleven genes mutated in BBS patients have been identified, and more are expected to exist, since about 20–30% of all families cannot be explained by the known loci. To investigate the etiopathogenesis of BBS, we created a mouse null for one of the murine homologues, Bbs4, to assess the contribution of one gene to the pleiotropic murine Bbs phenotype. Bbs4 null mice, although initially runted compared to their littermates, ultimately become obese in a gender-dependent manner, females earlier and with more severity than males. Blood chemistry tests indicated abnormal lipid profiles, signs of liver dysfunction, and elevated insulin and leptin levels reminiscent of metabolic syndrome. As in patients with BBS, we found age-dependent retinal dystrophy. Behavioral assessment revealed that mutant mice displayed more anxiety-related responses and reduced social dominance. We noted the rare occurrence of birth defects, including neural tube defects and hydrometrocolpos, in the null mice. Evaluations of these null mice have uncovered phenotypic features with age-dependent penetrance and variable expressivity, partially recapitulating the human BBS phenotype.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

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.     

Porphyrin-based Photocatalytic Nanolithography: A NEW FABRICATION TOOL FOR PROTEIN ARRAYS*

Nanoarray fabrication is a multidisciplinary endeavor encompassing materials science, chemical engineering, and biology. We formed nanoarrays via a new technique, porphyrin-based photocatalytic nanolithography. The nanoarrays, with controlled features as small as 200 nm, exhibited regularly ordered patterns and may be appropriate for (a) rapid and parallel proteomics screening of immobilized biomolecules, (b) protein-protein interactions, and/or (c) biophysical and molecular biology studies involving spatially dictated ligand placement. We demonstrated protein immobilization utilizing nanoarrays fabricated via photocatalytic nanolithography on silicon substrates where the immobilized proteins are surrounded by a non-fouling polymer background.Biomolecular arrays facilitate molecular aggregate investigation and high throughput analysis of immobilized biomolecules. Current biomolecular arraying capabilities are limited by relatively large sample volumes (typical spot sizes are on the order of ∼100–200 μm) and relatively long incubation times (1). Despite their limitations, protein microarray applications include autoantibody profiling, antibody response profiling, and identification and detection of bacterial and protein analytes as well as disease proteomics (oncoproteomics) (2–5). Typically a specific analyte is bound and detected by fluorescence, resulting in an expression profile or protein atlas.Nanoarrays are expected to expand the use of biomolecular arrays beyond drug discovery, medical diagnostics, and genetic testing to include point-of-care and in-the-field applications (6). We present a rapid and low cost photocatalytic lithography method for generating biomolecular nanoarrays on a non-fouling background appropriate for analysis of immobilized biomolecules.A number of publications have reviewed approaches to nanofabrication and bionanopatterning (7–9) of substrates including photolithography, contact printing (9, 10), imprint lithography (11–13), dip-pen lithography (14–17), and block co-polymer formation (18, 19). These approaches exhibit varying degrees of efficiency and success. Photocatalytic lithography (PCL)¹ is a more recently described technique that is capable of generating arrays. Lee and Sung (20) patterned silane layers by activating TiO₂ (a photocatalytic semiconductor) via 2-min exposure to UV light. They produced ∼600-nm parallel lines with 400-nm spaces, and they subsequently performed atomic layer deposition of zirconium dioxide (ZrO₂) onto exposed silanol groups (alternating octadecylsiloxane regions did not have necessary precursor molecules). The subsequent decomposition of alkylsiloxane monolayers with TiO₂ was reported to be 20 times faster than under UV irradiation in air.Previously we have described patterning via porphyrin-based PCL (21). Patterning is achieved within 10 s and with extremely low energy sources (an LED flashlight is sufficient). As presented below, we have miniaturized features patterned with this new technique and are now able to pattern on the scale of 200 nm; this is roughly half the reported scale achieved with PCL using photocatalytic semiconductors. We refer to patterning on this scale as porphyrin-based photocatalytic nanolithography (PCNL). We describe the implementation of porphyrin-based PCNL to form large scale nanoarrays appropriate for rapid, parallel, quantitative proteomics screening of immobilized biomolecules and to form spatially dictated ligand arrays for functional proteomics studies.