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+.     

Basic proline-rich proteins from human parotid saliva: relationships of the covalent structures of ten proteins from a single individual.

Eleven basic proline-rich proteins were purified from the parotid saliva of a single individual. The complete amino acid sequences of six of these were determined by conventional protein sequence methodology, bringing to nine the number of known primary structures of nonglycosylated basic proline-rich proteins from the same individual. The partial sequence of one additional protein is also reported. All of the basic proline-rich proteins studied contain segments with identical or very similar sequences, but with two possible exceptions, none of the proteins is derived from another secreted proline-rich protein. The amino acid sequences of nine nonglycosylated basic proline-rich proteins were compared with primary structures deduced from published nucleotide sequences of DNA coding for human parotid proline-rich proteins. The sequences align well, in general, but differences also exist pointing to the complexity of the genetics of these proteins. Seven secretory basic proline-rich proteins appear to be formed from three larger precursors by selective posttranslational proteolyses of arginyl bonds. One of the basic proline-rich proteins appears to derive from human acidic proline-rich proteins. The remaining two proteins studied do not conform to any DNA structure as yet reported. Two of the basic proline-rich proteins studied are phosphoproteins and exhibit abilities to inhibit hydroxyapatite formation in vitro.     

Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases

During macroautophagy/autophagy, mammalian Atg8-family proteins undergo 2 proteolytic processing events. The first exposes a COOH-terminal glycine used in the conjugation of these proteins to lipids on the phagophore, the precursor to the autophagosome, whereas the second releases the lipid. The ATG4 family of proteases drives both cleavages, but how ATG4 proteins distinguish between soluble and lipid-anchored Atg8 proteins is not well understood. In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates. Thus, while ATG4B is orders of magnitude faster at processing a soluble unprimed protein, all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates. The recognition of lipidated but not soluble substrates is sensitive to a COOH-terminal LIR motif both in vitro and in cells. We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs.     

Green‐wave surfing increases fat gain in a migratory ungulate

Each spring, migratory herbivores around the world track or ‘surf’ green waves of newly emergent vegetation to distant summer or wet‐season ranges. This foraging tactic may help explain the great abundance of migratory herbivores on many seasonal landscapes. However, the underlying fitness benefits of this life‐history strategy remain poorly understood. A fundamental prediction of the green‐wave hypothesis is that migratory herbivores obtain fitness benefits from surfing waves of newly emergent vegetation more closely than their resident counterparts. Here we evaluate whether this behavior increases body‐fat levels – a critically important correlate of reproduction and survival for most ungulates – in elk Cervus elaphus of the Greater Yellowstone Ecosystem. Using satellite imagery and GPS tracking data, we found evidence that migrants (n = 23) indeed surfed the green wave, occupying sites 12.7 days closer to peak green‐up than residents (n = 16). Importantly, individual variation in surfing may help account for up to 6 kg of variation in autumn body‐fat levels. Our findings point to a pathway for anthropogenic changes to the green wave (e.g. climate change) or migrants’ ability to surf it (e.g. development) to impact migratory populations. To explore this possibility, we evaluated potential population‐level consequences of constrained surfing with a heuristic model. If green‐wave surfing deteriorates by 5–15 days from observed, our model predicts up to a 20% decrease in pregnancy rates, a 2.5% decrease in population growth, and a 30% decrease in abundance over 50 years. By linking green‐wave surfing to fitness and illustrating potential effects on population growth, our study provides new insights into the evolution of migratory behavior and the prospects for the persistence of migratory ungulate populations in a changing world.     

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.     

Fire in the Brazilian Amazon: 1. Biomass,nutrient pools,and losses in slashed primary forests

Deforestation in the Brazilian Amazon has resulted in the conversion of >230,000 km² of tropical forest, yet little is known on the quantities of biomass consumed or the losses of nutrients from the ecosystem. We quantified the above-ground biomass, nutrient pools and the effects of biomass burning in four slashed primary tropical moist forests in the Brazilian Amazon. Total above-ground biomass (TAGB) ranged from 292 Mg ha^-1 to 436 Mg ha^-1. Coarse wood debris (>20.5 cm diameter) was the dominant fuel component. However, structure of the four sites were variable. Coarse wood debris comprised from 44% to 69% of the TAGB, while the forest floor (litter and rootmat) comprised from 3.7 to 8.0% of the TAGB. Total biomass consumption ranged from 42% to 57%. Fires resulted in the consumption of >99% of the litter and rootmat, yet <50% of the coarse wood debirs. Dramatic losses in C, N, and S were quantified. Lesser quantities of P, K, and Ca were lost by combustion processes. Carbon losses from the ecosystem were 58–112 Mg ha^-1. Nitrogen losses ranged from 817 to 1605 kg ha^-1 and S losses ranged from 92 to 122 kg ha^-1. This represents losses that are as high as 56%, 68%, and 49% of the total above-ground pools of these nutrients, respectively. Losses of P were as high as 20 kg ha^-1 or 32% of the above-ground pool. Losses to the atmosphere arising from primary slash fires were variable among sites due to site differences in concentration, fuel biomass, and fuel structure, climatic fluctuations, and anthropogenic influences. Compared to fires in other forest ecosystems, fires in slashed primary tropical evergreen forests result in among the highest total losses of nutrients ever measured. In addition, the proportion of the total nutrient pool lost from slash fires is higher in this ecosystem compared to other ecosystems due to a higher percentage of nutrients stored in above-ground biomass.     

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.      

Novel thrombin inhibitors that are based on a macrocyclic tripeptide motif

A series of macrocyclic -keto amides containing the D-Phe-Pro-Arg (fPR) motif were synthesized and evaluated in vitro as inhibitors of human -thrombin and bovine trypsin. Structure-function studies, relating ring size and modifications at the P3 and P1' positions to enzyme inhibition, are described. An X-ray crystallo-graphic study was performed on a ternary complex formed from 3i, thrombin, and hirugen.     

XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells

Previous work has shown that the three‐dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT‐185, KPT‐330/selinexor, and KPT‐8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment‐naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non‐lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711–2719, 2016. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.     

Carbon stocks of mangroves and losses arising from their conversion to cattle pastures in the Pantanos de Centla,Mexico

The conservation of mangroves and other coastal “blue carbon” ecosystems is receiving heightened attention because of recognition of their high ecosystem carbon stocks as well as vast areas undergoing land conversion. However, few studies have paired intact mangroves with degraded sites to determine carbon losses due to land conversion. To address this gap we quantified total ecosystem carbon stocks in mangroves and cattle pastures formed from mangroves in the large wetland complex of the Pantanos de Centla in SE Mexico. The mean total ecosystem carbon stocks of fringe and estuarine tall mangroves was 1358 Mg C/ha. In contrast the mean carbon stocks of cattle pastures was 458 Mg C/ha. Based upon a biomass equivalence of losses from the top 1 m of mangrove soils, the losses in carbon stocks from mangrove conversion are conservatively estimated at 1464 Mg CO₂e/ha. These losses were 7-fold that of emissions from tropical dry forest to pasture conversion and 3-fold greater than emissions from Amazon forest to pasture conversion. However, we found that limiting ecosystem carbon stocks differences to the surface 1 m or even 2 m soil depth will miss losses that occurred from deeper horizons. Mangrove conversion to other land uses comes at a great cost in terms of greenhouse gas emissions as well losses of other important ecosystem services.