An integrated approach to estimate how much urban afforestation can contribute to move towards carbon neutrality.

Urban afforestation is considered a promising nature-climate solution that may contribute to achieve climate neutrality by 2050, since it can increase C-storage and C-sequestration, whilst providing further multiple ecosystem services for citizens. However, the quantification of the CO2 sequestration capacity that may be provided by an urban forest as well as the capacity to impact the city-level C-balance and offset anthropogenic emissions is a complex issue. Methodological approaches, quantity and quality of information contained in urban tree database, and the level of detail of the planned urban forest can strongly influence the estimation of C-sequestration potential offered by urban forests. In this work, an integrated framework based on emission inventory, tree species/morphology and ecosystem modelling has been proposed for the city of Prato, Italy, a representative medium size European city to: i) evaluate the current C-sequestration capacity of urban trees; ii) upscale such capacity with different afforestation scenarios, iii) compare the sink capacity offered by ecosystems with current and projected anthropogenic emissions. Results indicated that the green areas within the Municipality of Prato can sequester 33.1 ktCO2 yr-1 under actual conditions and 51.0 ktCO2 yr-1 under the afforestation scenario which maximize the CO2 sequestration capacity, offsetting the 7.1 % and 11 % of the total emissions (465.8 ktCO2 yr-1), respectively. This study proves that, in the various afforestation scenarios tested, the contribution of urban afforestation to the municipality carbon balance is negligible and that carbon neutrality can only be reached by the substantial decarbonization of emission sectors.

Evaluation of anticoagulant activity of two algal polysaccharides.

Marine algae are important sources of phycocolloids like agar, carrageenans and alginates used in industrial applications. Algal polysaccharides have emerged as an important class of bioactive products showing interesting properties. The aim of our study was to evaluate the potential uses as anticoagulant drugs of algal sulphate polysaccharides extracted from Ulva fasciata (Chlorophyta) and Agardhiella subulata (Rhodophyta) collected in Ganzirri Lake (Cape Peloro Lagoon, north-eastern Sicily, Italy). Toxicity of algal extracts through trypan blue test and anticoagulant action measured by activated partial thromboplastin time (APTT), prothrombin time (PT) test has been evaluated. Algal extracts showed to prolong the PT and APTT during the coagulation cascade and to avoid the blood coagulation of samples. Furthermore, the algal extracts lack toxic effects towards cellular metabolism and their productions are relatively at low cost. This permits to consider the algae as the biological source of the future.

In vitro evaluation of antibacterial activity of Asparagopsis taxiformis from the Straits of Messina against pathogens relevant in aquaculture.

Ethanol extracts of Asparagopsis taxiformis collected from the Straits of Messina (Italy) were screened for antibacterial activity against pathogenic shellfish and fish bacteria previously isolated from local marine and brackish environments. Genetic labelling by DNA barcoding allowed us to identify the algal population as a biogeographical strain conspecific to A. taxiformis. The extract obtained in May showed the broadest antibacterial activity against all tested pathogenic bacteria, especially against Vibrio alginolyticus, Vibrio vulnificus and Aeromonas salmonicida subsp. salmonicida. Moderate activity was observed against Photobacterium damselae subsp. damselae and Photobacterium damselae subsp. piscicida, Salmonella sp., Vibrio cholerae, Vibrio harveyi and Vibrio parahaemolyticus. The absence of cytotoxic effects of active algal extracts was verified using trypan blue exclusion test on cells of digestive glands of Mytilus galloprovincialis. The results indicated that ethanol extracts of A. taxiformis could represent a source of antibacterial substances with potential use in aquaculture.

Soluble beta-amyloid1-40 induces NMDA-dependent degradation of postsynaptic density-95 at glutamatergic synapses.

Amyloid-beta (Abeta) has been implicated in memory loss and disruption of synaptic plasticity observed in early-stage Alzheimer's disease. Recently, it has been shown that soluble Abeta oligomers target synapses in cultured rat hippocampal neurons, suggesting a direct role of Abeta in the regulation of synaptic structure and function. Postsynaptic density-95 (PSD-95) is a postsynaptic scaffolding protein that plays a critical role in synaptic plasticity and the stabilization of AMPA (AMPARs) and NMDA (NMDARs) receptors at synapses. Here, we show that exposure of cultured cortical neurons to soluble oligomers of Abeta(1-40) reduces PSD-95 protein levels in a dose- and time-dependent manner and that the Abeta1(1-40)-dependent decrease in PSD-95 requires NMDAR activity. We also show that the decrease in PSD-95 requires cyclin-dependent kinase 5 activity and involves the proteasome pathway. Immunostaining analysis of cortical cultured neurons revealed that Abeta treatment induces concomitant decreases in PSD-95 at synapses and in the surface expression of the AMPAR glutamate receptor subunit 2. Together, these data suggest a novel pathway by which Abeta triggers synaptic dysfunction, namely, by altering the molecular composition of glutamatergic synapses.

Glutamate-induced delta-catenin redistribution and dissociation from postsynaptic receptor complexes.

Delta-catenin (or neural plakophilin-related arm-repeat protein/neurojungin) is primarily a brain specific member of the p120(ctn) subfamily of armadillo/beta-catenin proteins that play important roles in neuronal development. Our previous studies have shown that the ectopic expression of delta-catenin induces the formation of dendrite-like extensions and that the overexpression of delta-catenin promotes dendritic branching and increases spine density. Here we demonstrate that delta-catenin displays a dendritic distribution pattern in the adult mouse brain and is co-enriched with postsynaptic density-95 (PSD-95) in the detergent insoluble postsynaptic scaffolds. Delta-catenin forms stable complexes with excitatory neurotransmitter receptors including ionotropic N-methyl-D-aspartic acid receptor 2A (NR2A), metabotropic glutamate receptor 1alpha (mGluR1alpha), as well as PSD-95 in vivo. In cultured primary embryonic neurons, delta-catenin clusters co-distribute with filamentous actin and resist detergent extraction. In dissociated hippocampal neurons overexpressing delta-catenin, glutamate stimulation leads to a rapid redistribution of delta-catenin that can be attenuated by 6-cyano-7-nitroquinoxaline-2,3-dione and dizocilpine, selective inhibitors of ionotropic glutamate receptors. Upon glutamate receptor activation, delta-catenin becomes down-regulated and its association with NR2A and mGluR1alpha in cultured neurons is diminished. These findings support a possible functional connection between delta-catenin and the glutamatergic excitatory synaptic signaling pathway during neuronal development.

Expression and characterization of Flag-epitope- and hexahistidine-tagged derivatives of saxiphilin for use in detection and assay of saxitoxin.

Saxiphilin is a plasma protein from the bullfrog (Rana catesbiana) that binds saxitoxin (STX), a causative agent of paralytic shellfish poisoning. Saxiphilin is homologous to transferrin and consists of two internally homologous domains called the N-lobe and the C-lobe. STX binds to a single site in the C-lobe of saxiphilin. In this study, cloned genes coding for recombinant saxiphilin and C-lobe saxiphilin were modified to contain two tandemly located affinity tags, Flag epitope (DYKDDDDK) and His(6) (HHHHHH), at the protein C-terminus and were expressed in cultured insect cells using baculovirus vectors. Both tagged proteins are readily detected on immunoblots by anti-Flag monoclonal antibody. Flag-His(6)-tagged saxiphilin was purified to homogeneity using Ni(2+)-chelate affinity chromatography and Heparin Sepharose chromatography. Equilibrium analysis of [3H]STX binding to tagged saxiphilin and tagged C-lobe saxiphilin gave K(D) values of 0.75 and 2.7 nM, respectively. Flag-His(6)-tagged saxiphilin was also utilized in a microtiter well solid-phase assay with Reacti-bind metal chelate plates to measure [3H]STX binding and binding competition by unlabeled STX. Such Flag-His(6)-tagged derivatives of saxiphilin have many possible applications in the assay of STX and related toxinological research.

NUDEL is a novel Cdk5 substrate that associates with LIS1 and cytoplasmic dynein.

Disruption of one allele of the LIS1 gene causes a severe developmental brain abnormality, type I lissencephaly. In Aspergillus nidulans, the LIS1 homolog, NUDF, and cytoplasmic dynein are genetically linked and regulate nuclear movements during hyphal growth. Recently, we demonstrated that mammalian LIS1 regulates dynein functions. Here we characterize NUDEL, a novel LIS1-interacting protein with sequence homology to gene products also implicated in nuclear distribution in fungi. Like LIS1, NUDEL is robustly expressed in brain, enriched at centrosomes and neuronal growth cones, and interacts with cytoplasmic dynein. Furthermore, NUDEL is a substrate of Cdk5, a kinase known to be critical during neuronal migration. Inhibition of Cdk5 modifies NUDEL distribution in neurons and affects neuritic morphology. Our findings point to cross-talk between two prominent pathways that regulate neuronal migration.

D2 dopamine antisense RNA expression vector, unlike haloperidol, produces long-term inhibition of D2 dopamine-mediated behaviors without causing Up-regulation of D2 dopamine receptors.

Long-term inhibition of D2 dopamine receptors using classic D2 dopamine receptor antagonists such as haloperidol often causes a compensatory up-regulation of the D2 dopamine receptors. We investigated whether the long-term inhibition of D2 dopamine receptors using an eukaryotic expression vector housing a cDNA sequence encoding an antisense RNA directed to the D2 dopamine receptor transcript (D2 antisense vector) would also produce up-regulation of the D2 receptors. Single, bilateral injections of the D2 antisense vector into the corpora striata of mice inhibited the stereotypy induced by acute challenge injections with the D2/D3 dopamine receptor agonist quinpirole but did not inhibit the grooming induced by acute challenge injections with the D1 agonist SKF 38393. Similar treatment with the D2 antisense vector produced a long-term (>1 month) cataleptic response without producing tolerance to challenge injections with haloperidol. By contrast, catalepsy induced by a single injection of haloperidol lasted only approximately 2 days, and tolerance developed to its effects after long-term treatment. Repeated treatment of mice with haloperidol resulted in an inhibition of apomorphine-induced climbing behavior throughout the time of treatment with haloperidol, but the climbing behavior markedly increased to levels significantly higher than that of the control mice immediately after withdrawal from haloperidol treatment. This increased climbing was accompanied by increased levels of D2 dopamine receptors in the striatum. By contrast, single, bilateral intrastriatal injections of the D2 antisense vector significantly inhibited apomorphine-induced climbing for approximately 30 days but failed to increase the climbing behavior or the levels of D2 dopamine receptors in striatum over those of the control values. These results suggest that a single injection of a D2 antisense RNA expression vector into mouse striatum produces specific, long-term inhibition of D2 dopamine receptor behaviors without causing a compensatory increase in the levels or function of D2 dopamine receptors.

Expression of a D2 dopamine receptor antisense RNA in brain inhibits D2-mediated behaviors.

Drugs currently used to treat disorders of dopamine-mediated behaviors in the central nervous system are non-selective in that they interact not only with more than one isoform of dopamine receptor but also with receptors for other neurotransmitters. A new strategy to inhibit the actions of individual dopamine receptor subtypes is to inhibit the synthesis of the receptors through the use of oligonucleotides antisense to the transcripts encoding the different receptors. Earlier studies showed that oligodeoxynucleotides antisense to the D1 or D2 dopamine receptor messenger RNAs specifically inhibited the biological actions mediated by these individual isoforms of the dopamine receptor. However, these actions were relatively short-lasting. To determine whether one can achieve long-lasting inhibition of dopamine responses, while still taking advantage of the highly selective nature of an antisense strategy, an expression vector was employed that generates antisense RNA to the transcript encoding the D2 dopamine receptor. A single intrastriatal injection of this vector generated an antisense RNA to the D2 dopamine receptor, selectively reduced the levels of D2 dopamine receptors, and caused selective, long-term inhibition of behaviors mediated by D2 dopamine agonists. Such an antisense RNA strategy may find use in studying the function of dopaminergic receptors and in disorders associated with dopaminergic hyperactivity.

An evolutionarily conserved binding site for serine proteinase inhibitors in large conductance calcium-activated potassium channels.

Complementary DNA coding for the channel-forming alpha-subunit of a large conductance Ca(2+)-activated K+ channel (maxi Kca channel) was cloned from bovine aortic smooth muscle cells. This cloned mammalian KCa channel (Bslo) and its homolog from Drosophila (Dslo) were expressed in the HEK293 human embryonic kidney cell line. Both Bslo and Dslo KCa channels were sensitive to inhibition by the internally applied serine proteinase inhibitors: bovine pancreatic trypsin inhibitor (BPTI, KD = 7.0 microM for Bslo and 2.6 microM for Dslo) and chicken ovoinhibitor (OI, KD = 1.5 microM for Bslo and 11.4 microM for Dslo). BPTI and OI are members of the Kunitz and Kazal families of proteinase inhibitors, respectively. The approximately 60-residue inhibitory domains of these proteins have a different tertiary structure except in the region of a loop formed by approximately 6 residues, in which the peptide backbone adopts a similar conformation complementary to the active site cleft of many serine proteinases. At the single-channel level, BPTI and OI were found to inhibit KCa channels by a similar mechanism involving the production of discrete low-conductance events. These two inhibitors also exhibited competitive behavior, suggesting that they bind to an overlapping site. Kinetic characterization revealed that the dissociation rate of BPTI from the bovine KCa channel is fast (k(off) = 0.41 s-1), whereas that from the Drosophila KCa channel is slow (k(off) = 9.0 x 10(-4) s-1) and indicative of a strong molecular interaction. The stable complex of BPTI and trypsin was inactive as a KCa channel inhibitor, further supporting the idea that the trypsin inhibitory loop of BPTI recognizes a specific site on the channel protein. These results lead to the conclusion that the alpha-subunit of maxi KCa channels contains a conserved proteinase inhibitor binding site. We hypothesize that this site corresponds to a C-terminal domain of the channel protein that structurally resembles serine proteinases.

The divergent homeobox gene PBX1 is expressed in the postnatal subventricular zone and interneurons of the olfactory bulb.

In the mammalian brain, an important phase of neurogenesis occurs postnatally in the subventricular zone (SVZ). This region consists of a heterogeneous population of cells, some mitotically active, others postmitotic. A subset of mitotically active SVZ precursor cells gives rise to a population of neurons that migrates over a long distance to their final destination, the olfactory bulb. Other SVZ precursor cells continue to proliferate or undergo cell death. The combination of genes that regulates proliferation and cell fate determination of SVZ precursor cells remains to be identified. We have used the rat homolog of the human homeobox gene PBX1 in Northern analysis and in situ hybridization studies to determine the temporal and regional localization of PBX1 expression during embryonic and postnatal rat brain development. PBX1 is expressed embryonically in the telencephalon. In addition, it is expressed at high levels postnatally in the SVZ, in the migratory pathway to the olfactory bulb, and in the layers of the olfactory bulb that are the targets of these migratory neurons. Combining in situ hybridization for PBX1 with immunostaining for markers of cell proliferation (PCNA), postmitotic neurons (class III beta-tubulin), and glia (GFAP), we show that SVZ proliferating cells and their neuronal progeny express rat PBX1 mRNA, whereas glial cells do not express detectable levels of PBX1. The expression of PBX1 in SVZ precursor cells and postmitotic neurons suggests a role for PBX1 in the generation of olfactory bulb interneurons and in mammalian neurogenesis.

Uptake and distribution of fluorescein-labeled D2 dopamine receptor antisense oligodeoxynucleotide in mouse brain.

To determine the uptake and distribution of oligodeoxynucleotides in brain, a 20-mer phosphorothioated oligodeoxynucleotide complementary to a portion of the D2 dopamine receptor mRNA was fluorescently labeled with fluorescein isothiocyanate (FITC) and injected into the lateral cerebral ventricles of mice. At various survival times after the injection, the brains were removed, fixed, sectioned, and viewed under a fluorescent microscope. The results showed that the oligodeoxynucleotide was rapidly taken up into the brain. Initially the label was relatively diffusely spread throughout the interstitial spaces of the brain, then became redistributed to the cellular compartments. The signal extended from those forebrain nuclei located immediately in contact with the ventricles, such as the corpus striatum, septum, and hippocampus, to areas further removed from the ventricles, such as the cerebral cortex, nucleus accumbens, and substantia nigra. When the FITC-labeled D2 antisense oligodeoxynucleotide was given once daily for 4 d, the signal intensity seen 24 h after the last injection appeared to be of greater intensity overall compared to that seen after a single injection. At early time-points the oligodeoxynucleotide signals appeared to be punctuated and were found in cell bodies as well as in proximal dendritic processes. However, not all cells were equally labeled, suggesting an uneven uptake and accumulation of the D2 antisense into the various cell types. At later time-points the fluorescent signal appeared granular; at these times the injected material was largely degraded. These studies show that a D2 dopamine receptor antisense oligodeoxynucleotide is rapidly taken up from cerebral ventricles into brain, becomes widely distributed throughout the brain tissue to areas far removed from direct contact with the ventricles, and appears to accumulate to a different extent in the various brain areas and cell types.

Expression of saxiphilin in insect cells and localization of the saxitoxin-binding site to the C-terminal domain homologous to the C-lobe of transferrins.

Saxiphilin is a plasma protein from the bullfrog (Rana catesbeiana) that is homologous to transferrin. Most known transferrins contain two binding sites for Fe3+/HCO3-, one in each of two homologous domains called the N-lobe and C-lobe. However, native saxiphilin does not bind Fe3+ but stoichiometrically binds one molecule of the neurotoxin saxitoxin (STX) with a dissociation constant (KD) of approximately 0.2 nM. To pursue structural analysis of the STX binding sites, cDNA encoding saxiphilin was used to construct a baculovirus expression vector that directs synthesis and secretion of a approximately 92-kDa recombinant saxiphilin protein (R-sax) in cultured insect cells. Culture medium harvested from infected cells contained 25-67 pmol of [3H]STX binding sites/mL with a KD of 0.22 nM. The kinetics and pH dependence (pK0.5 = 5.4) of [3H]STX binding to R-sax are similar to native saxiphilin, implying proper folding and functional activity. Another baculovirus expression vector was constructed to encode a deletion mutant of saxiphilin consisting of the first 20 N-terminal residues containing the secretory signal sequence spliced to the C-terminal, 361-residue fragment homologous to the C-lobe domain of transferrins. This vector directed the secretion of a approximately 38-kDa derivative of saxiphilin (C-sax) that was recognized by antisaxiphilin antibody. C-sax also exhibited [3H]STX binding activity with a lower affinity KD of approximately 0.9 nM, a 4-fold faster dissociation rate for [3H]STX than native saxiphilin, and a pH dependence (pK0.5 = 5.7) similar to R-sax (pK0.5 = 5.4).(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of bullfrog saxiphilin: a unique relative of the transferrin family that binds saxitoxin.

Plasma and tissue of certain vertebrates contain a protein called saxiphilin that specifically binds the neurotoxin saxitoxin with nanomolar affinity. We describe the isolation of a cDNA clone of saxiphilin from liver of the North American bullfrog (Rana catesbeiana). The cDNA sequence encodes a protein that is evolutionarily related to members of the transferrin family of Fe(3+)-binding proteins. Pairwise sequence alignment of saxiphilin with various transferrins reveals amino acid identity as high as 51% and predicts 14 disulfide bonds that are highly conserved. The larger size of saxiphilin (91 kDa) versus serum transferrin (approximately 78 kDa) is primarily due to a unique insertion of 144 residues. This insertion contains a 49-residue domain classified as a type 1 repetitive element of thyroglobulin, which is shared by a variety of membrane, secreted, and extracellular matrix proteins. Saxiphilin also differs from transferrins in 9 of 10 highly conserved amino acids in the two homologous Fe3+/HCO3-binding sites of transferrin. Identification of saxiphilin implies that transferrin-like proteins comprise a diverse superfamily with functions other than iron binding.