Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.     

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

Identifying the minimal copper- and zinc-binding site sequence in amyloid-beta peptides

With a combination of complementary experimental techniques, namely sedimentation assay, Fourier transform infrared spectroscopy, and x-ray absorption spectroscopy, we are able to determine the atomic structure around the metal-binding site in samples where amyloid-beta (Abeta) peptides are complexed with either Cu(II) or Zn(II). Exploiting information obtained on a selected set of fragments of the Abeta peptide, we identify along the sequence the histidine residues coordinated to the metal in the various peptides we have studied (Abeta(1-40), Abeta(1-16), Abeta(1-28), Abeta(5-23), and Abeta(17-40)). Our data can be consistently interpreted assuming that all of the peptides encompassing the minimal 1-16 amino acidic sequence display a copper coordination mode that involves three histidines (His(6), His(13), and His(14)). In zinc-Abeta complexes, despite the fact that the metal coordination appears to be more sensitive to solution condition and shows a less rigid geometry around the binding site, a four-histidine coordination mode is seen to be preferred. Lacking a fourth histidine along the Abeta peptide sequence, this geometrical arrangement hints at a Zn(II)-promoted interpeptide aggregation mode.     

Post-translational changes of chromosomal proteins in rat cerebellum during postnatal development

Acetylation, phosphorylation and methylation of nuclear proteins in rat cerebellum at 10 and 30 days of age were investigated in vitro. Isolated nuclei were incubated in the presence of [1-14C]acetyl CoA, S-adenosyl [methyl-3H]methionine and [gamma-32P]ATP and then separated into histones and non histone proteins (NHP), which were further fractionated by polyacrylamide gel electrophoresis. The results obtained indicate that acetylation, phosphorylation and methylation of both basic and acidic proteins decrease from 10 to 30 days of age. Electrophoretic analysis of histones shows that the decrease mainly concerns H1, H3, and H2b fractions. The H3 fraction is always more labeled than the other fractions and shows the major changes during postnatal development. Phosphorylation of H2a and H4 fractions increases from 10 to 30 days of age, whereas acetylation and methylation of these fractions do not show significant changes from 10 to 30 days. The densitometric and radioactive patterns of NHP show considerable changes between 10 and 30 days, especially in the high molecular weight region. The incorporation of 14C-acetyl and 3H-methyl groups and of 32P phosphate appears to be generalized throughout the molecular weight range and decreases from 10 to 30 days of age. The methylation of an as yet unidentified protein with a molecular weight of approximately 110,000 daltons occurred at both ages.     

Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex

Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.     

Chromatographic behaviour of the molecular forms of guinea-pig skeletal muscle cytoplasmic malate dehydrogenase

The isolated molecular forms of guinea-pig skeletal muscle cytoplasmic malate dehydrogenase have a different chromatographic behaviour through affinity or hydrophobic interaction gels; in all cases the retention of the B form is more noticeable. Chromatography of a partly purified preparation through 5' AMP-Sepharose allows both molecular forms of malate dehydrogenase to be separated and obtained free from lactate dehydrogenase.     

Inactivation of Tgfbr2 in Osterix-Cre expressing dental mesenchyme disrupts molar root formation

It has been difficult to examine the role of TGF-ß in post-natal tooth development due to perinatal lethality in many of the signaling deficient mouse models. To address the role of Tgfbr2 in postnatal tooth development, we generated a mouse in which Tgfbr2 was deleted in odontoblast- and bone-producing mesenchyme. Osx-Cre;Tgfbr2^fl/fl mice were generated (Tgfbr2^cko) and post-natal tooth development was compared in Tgfbr2^cko and control littermates. X-ray and μCT analysis showed that in Tgfbr2^cko mice radicular dentin matrix density was reduced in the molars. Molar shape was abnormal and molar eruption was delayed in the mutant mice. Most significantly, defects in root formation, including failure of the root to elongate, were observed by postnatal day 10. Immunostaining for Keratin-14 (K14) was used to delineate Hertwig's epithelial root sheath (HERS). The results showed a delay in elongation and disorganization of the HERS in Tgfbr2^cko mice. In addition, the HERS was maintained and the break up into epithelial rests was attenuated suggesting that Tgfbr2 acts on dental mesenchyme to indirectly regulate the formation and maintenance of the HERS. Altered odontoblast organization and reduced Dspp expression indicated that odontoblast differentiation was disrupted in the mutant mice likely contributing to the defect in root formation. Nevertheless, expression of Nfic, a key mesenchymal regulator of root development, was similar in Tgfbr2^cko mice and controls. The number of osteoclasts in the bone surrounding the tooth was reduced and osteoblast differentiation was disrupted likely contributing to both root and eruption defects. We conclude that Tgfbr2 in dental mesenchyme and bone is required for tooth development particularly root formation.     

Canopy attachment position influences metabolism and peel constituency of European pear fruit

Background

Inconsistent pear fruit ripening resulting from variable harvest maturity within tree canopies can contribute to postharvest losses through senescence and spoilage that would otherwise be effectively managed using crop protectant and storage regimes. Because those inconsistencies are likely based on metabolic differences, non-targeted metabolic profiling peel of ‘d’Anjou’ pears harvested from the external or internal canopy was used to determine the breadth of difference and link metabolites with canopy position during long-term controlled atmosphere storage.

Results

Differences were widespread, encompassing everything from expected distinctions in flavonol glycoside levels between peel of fruit from external and internal canopy positions to increased aroma volatile production and sucrose hydrolysis with ripening. Some of the most substantial differences were in levels of triterpene and phenolic peel cuticle components among which acyl esters of ursolic acid and fatty acyl esters of p-coumaryl alcohol were higher in the cuticle of fruit from external tree positions, and acyl esters of α-amyrin were elevated in peel of fruit from internal positions. Possibly the most substantial dissimilarities were those that were directly related to fruit quality. Phytosterol conjugates and sesquiterpenes related to elevated superficial scald risk were higher in pears from external positions which were to be potentially rendered unmarketable by superficial scald. Other metabolites associated with fruit aroma and flavor became more prevalent in external fruit peel as ripening progressed and, likewise, with differential soluble solids and ethylene levels, suggesting the final product not only ripens differentially but the final fruit quality following ripening is actually different based on the tree position.

Conclusions

Given the impact tree position appears to have on the most intrinsic aspects of ripening and quality, every supply chain management strategy would likely lead to diverse storage outcomes among fruit from most orchards, especially those with large canopies. Metabolites consistently associated with peel of fruit from a particular canopy position may provide targets for non-destructive pre-storage sorting used to reduce losses contributed by this inconsistency.

     

Monitoring tropical forests under a functional perspective with satellite‐based vegetation optical depth

Monitoring ecosystem functions in forests is a priority in a climate change scenario, as climate‐induced events may initially alter the functions more than slow‐changing attributes, such as biomass. The ecosystem functional properties (EFPs) are quantities that characterize key ecosystem processes. They can be derived by point observations of gas and energy exchanges between the ecosystems and the atmosphere that are collected globally at FLUXNET flux tower sites and upscaled at ecosystem level. The properties here considered describe the ability of ecosystems to optimize the use of resources for carbon uptake. They represent functional forest information, are dependent on environmental drivers, linked to leaf traits and forest structure, and influenced by climate change effects. The ability of vegetation optical depth (VOD) to provide forest functional information is investigated using 2011–2014 satellite data collected by the Soil Moisture and Ocean Salinity mission and using the EFPs as reference dataset. Tropical forests in Africa and South America were analyzed, also according to ecological homogeneous units. VOD jointly with water deficit information explained 93% and 87% of the yearly variability in both flux upscaled maximum gross primary productivity and light use efficiency functional properties, in Africa and South America forests respectively. Maps of the retrieved properties evidenced changes in forest functional responses linked to anomalous climate‐induced events during the study period. The findings indicate that VOD can support the flux upscaling process in the tropical range, affected by high uncertainty, and the detection of forest anomalous functional responses. Preliminary temporal analysis of VOD and EFP signals showed fine‐grained variability in periodicity, in signal dephasing, and in the strength of the relationships. In selected drier forest types, these satellite data could also support the monitoring of functional dynamics.