Diadenosine tetraphosphate (Ap<Subscript>4</Subscript>A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment

Reducing cell death during the secondary injury is a major priority in the development of a cure for traumatic spinal cord injury (SCI). One of the earliest processes that follow SCI is the excitotoxicity resulting from the massive release of excitotoxicity mediators, including ATP, which induce an excessive and/or prolonged activation of their receptors and a deregulation of the calcium homeostasis. Diadenosine tetraphosphate (Ap₄A) is an endogenous purinergic agonist, present in both extracellular and intracellular fluids, with promising cytoprotective effects in different diseases including neurodegenerative processes. In a search for efficient neuroprotective strategies for SCI, we have tested the capability of Ap₄A to reduce the excitotoxic death mediated by the ATP-induced deregulation of calcium homeostasis and its consequences on tissue preservation and functional recovery in a mouse model of moderate contusive SCI. Our analyses with the murine neural cell line Neuro2a demonstrate that treatment with Ap₄A reduces ATP-dependent excitotoxic death by both lowering the intracellular calcium response and decreasing the expression of specific purinergic receptors. Follow-up analyses in a mouse model of contusive SCI showed that acute administration of Ap₄A following SCI reduces tissue damage and improves motor function recovery. These results suggest that Ap₄A cytoprotection results from a decrease of the purinergic tone preventing the effects of a massive release of ATP after SCI, probably together with a direct induction of anti-apoptotic and pro-survival pathways via activation of P2Y₂ proposed in previous studies. In conclusion, Ap₄A may be a good candidate for an SCI therapy, particularly to reduce excitotoxicity in combination with other modulators and/or inhibitors of the excitotoxic process that are being tested.     

The subcommissural organ expresses D<Subscript>2</Subscript>, D<Subscript>3</Subscript>, D<Subscript>4</Subscript>, and D<Subscript>5</Subscript> dopamine receptors

Dopamine receptors have been found in certain populations of non-neuronal cells in the brain, viz., discrete areas of ciliated ependyma and the ependymal cells of the choroid plexus. We have studied the presence of both tyrosine-hydroxylase-immunoreactive nerve fibers and dopamine receptors in the subcommissural organ (SCO), an ependymal brain gland that is located in the roof of the third ventricle and that secretes, into the cerebrospinal fluid, glycoproteins that aggregate to form Reissners fiber (RF). Antibodies against D₂, D₃, D₄, and D₅ dopamine receptors were used in immunoblots of bovine striatum, fresh SCO, and organ-cultured SCO, and in immunocytochemistry of the bovine, rat, and mouse SCO. Only a few tyrosine-hydroxylase fibers appeared to reach the SCO. However, virtually all the secretory ependymal and hypendymal cells of the SCO immunoreacted with antibodies against D₂, D₄, and D₅ receptors, with the last-mentioned rendering the strongest reaction, especially at the ventricular cell pole of the secretory ependymocytes, suggesting that dopamine might reach the SCO via the cerebrospinal fluid. The antibodies against the four subtypes of receptors revealed corresponding bands in immunoblots of striatum and fresh SCO. Although the cultured SCO displayed dopamine receptors, dopamine had no apparent effect on the expression of the SCO-spondin gene/protein or on the release of RF-glycoproteins (SCO-spondin included) by SCO explants, suggesting that dopamine affects the function(s) of the SCO differently from the secretion of RF-glycoproteins.Financial support was provided by grants PI 030756 and Red CIEN, Instituto de Salud Carlos III, Spain (to J.M.P.F.), and 1030265 from Fondecyt, Chile (to E.M.R.)     

Immobilization of glucose oxidase on Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>/SiO<Subscript>2</Subscript> magnetic nanoparticles

Glucose oxidase (GOD) was covalently immobilized onto Fe₃O₄/SiO₂ magnetic nanoparticles (FSMNs) using glutaraldehyde (GA). Optimal immobilization was at pH 6 with 3-aminopropyltriethoxysilane at 2% (v/v), GA at 3% (v/v) and 0.143 g GOD per g carrier. The activity of immobilized GOD was 4,570 U/g at pH 7 and 50°C. The immobilized GOD retained 80% of its initial activity after 6 h at 45°C while free enzyme retained only 20% activity. The immobilized GOD maintained 60% of its initial activity after 6 cycles of repeated use and retained 75% of its initial activity after 1 month at 4°C whereas free enzymes retained 62% of its activity.     

Increased Vascular Resistance by Prostaglandins B<Subscript>1</Subscript> and B<Subscript>2</Subscript> in the Isolated Rat Pancreas

IN view of the possibility that prostaglandins (PG) regulate local blood flow¹, we are investigating this activity in the pancreas. We have already found that PGE₂ reduces vascular resistance in the perfused rat pancreas whereas PGF_2α has the opposite effect². These effects were seen at low doses (0.1 µg/ml.) and with good reproducibility.     

Synthesis of Protein-Inorganic Nanohybrids with Improved Catalytic Properties Using Co<Subscript>3</Subscript>(PO<Subscript>4</Subscript>)<Subscript>2</Subscript>

In the present study, a method for easy and rapid synthesis of lipase nanohybrids was evaluated using cobalt chloride as an encapsulating agent. The synthesized nanohybrids exhibited higher activity (181%) compared to free lipase and improved catalytic properties at higher temperature and in harsh conditions. The nanohybrids retained 84% of their residual activity at 25 °C after 10 days. In addition, these nanohybrids also exhibited high storage stability and reusability. Collectively, the synthesis of carrier-free immobilized biocatalysts was performed rapidly within 24 h at 4 °C. Their high reusability and catalytic activities highlight the broad applicability of this method for catalysis in organic and aqueous media.     

Activity of C<Subscript>4</Subscript> enzymes in C<Subscript>3</Subscript>-type herbaceous plants

The activity of enzymes characteristic for C₄-type photosynthesis was determined in different organs of two herbaceous plants: Reynoutria japonica Houtt. and Helianthus tuberosus L. The activity of phosphoenolpyruvate carboxylase (PEPC) was usually higher in the roots, some of the stem tissues and petioles in comparison to the leaf blades. The highest activity of malic enzymes (NAD-ME, NADP-ME) and phosphoenolpyruvate carboxykinase (PEPCK) was in the petioles and stem tissues of both plants and the lowest in the leaf blades and the pith of Helianthus tuberosus L.     

Synthesis and characterization of core-shell Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>-gold-chitosan nanostructure

Background  

Fe₃O₄-gold-chitosan core-shell nanostructure can be used in biotechnological and biomedical applications such as magnetic bioseparation, water and wastewater treatment, biodetection and bioimaging, drug delivery, and cancer treatment.     

Emissions of CO<Subscript>2</Subscript>, CH<Subscript>4</Subscript> and N<Subscript>2</Subscript>O from undisturbed,drained and mined peatlands in Estonia

The aim of this study is to estimate emissions of greenhouse gases CO₂, CH₄ and N₂O, and the effects of drainage and peat extraction on these processes, in Estonian transitional fens and ombrotrophic bogs. Closed-chamber-based sampling lasted from January to December 2009 in nine peatlands in Estonia, covering areas with different land-use practices: natural (four study sites), drained (six sites), abandoned peat mining (five sites) and active peat mining areas (five sites). Median values of soil CO₂ efflux were 1,509, 1,921, 2,845 and 1,741 kg CO₂-C ha^?1 year^?1 from natural, drained, abandoned and active mining areas, respectively. Emission of CH₄-C (median values) was 85.2, 23.7, 0.07 and 0.12 kg ha^?1 year^?1, and N₂O-N ?0.05, ?0.01, 0.18 and 0.19 kg ha^?1 year^?1, respectively. There were significantly higher emissions of CO₂ and N₂O from abandoned and active peat mining areas, whereas CH₄ emissions were significantly higher in natural and drained areas. Significant Spearman rank correlation was found between soil temperature and CO₂ flux at all sites, and CH₄ flux with high water level at natural and drained areas. Significant increase in CH₄ flux was detected for groundwater levels above 30 cm.     

Simulation and optical spectroscopy of a DC discharge in a CH<Subscript>4</Subscript>/H<Subscript>2</Subscript>/N<Subscript>2</Subscript> mixture during deposition of nanostructured carbon films

Two-dimensional numerical simulations of a dc discharge in a CH₄/H₂/N₂ mixture in the regime of deposition of nanostructured carbon films are carried out with account of the cathode electron beam effects. The distributions of the gas temperature and species number densities are calculated, and the main plasmachemical kinetic processes governing the distribution of methyl radicals above the substrate are analyzed. It is shown that the number density of methyl radicals above the substrate is several orders of magnitude higher than the number densities of other hydrocarbon radicals, which indicates that the former play a dominant role in the growth of nanostructured carbon films. The model is verified by comparing the measured optical emission profiles of the H(n ≡ 3), C ₂ ^* , CH*, and CN* species and the calculated number densities of excited species, as well as the measured and calculated values of the discharge voltage and heat fluxes onto the electrodes and reactor walls. The key role of ion–electron recombination and dissociative excitation of H₂, C₂H₂, CH₄, and HCN molecules in the generation of emitting species (first of all, in the cold regions adjacent to the electrodes) is revealed.     

Production of bioethanol from sugarcane bagasse using NH<Subscript>4</Subscript>OH-H<Subscript>2</Subscript>O<Subscript>2</Subscript> pretreatment and simultaneous saccharification and co-fermentation

In this study, we investigated the production of bioethanol from sugarcane bagasse (SCB) using an NH₄OH-H₂O₂ pretreatment and simultaneous saccharification and co-fermentation (SScF). Response surface methodology and a 2³ Box-Behnken design were used to evaluate the effect of different liquid mixture concentrations, liquid-to-solid ratios (LSRs) and pretreatment temperatures on the production of ethanol. The liquid mixture concentration and LSR significantly influenced the fermentation efficiency. Based on ridge max analysis, the following pretreatment conditions resulted in a fermentation efficiency of 95.79 ± 0.01%: liquid mixture concentration 53%, LSR 28, and a temperature of 63°C. A morphological analysis performed using scanning electron microscopy (SEM) and chemical characterization revealed that these pretreatment conditions were effective in disrupting the sugarcane fibers and removing lignin. Ethanol fermentation with the pretreated SCB using SScF in yeast SHY 07-1 resulted in an ethanol concentration of 14.65 ± 0.17 g/L, an ethanol yield of 0.48 ± 0.01 g/g, and an ethanol productivity of 0.12 ± 0.01 g/(L/h), which represents increases of 106.02, 89.98, and 107.02%, respectively, over the values obtained from SScF with untreated SCB.     

Effects of elevated atmospheric CO<Subscript>2</Subscript> on the nutritional ecology of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> grass-feeding caterpillars

It is plausible that the nutritional quality of C₃ plants will decline more under elevated atmospheric CO₂ than will the nutritional quality of C₄ plants, causing herbivorous insects to increase their feeding on C₃ plants relative to C₄ plants. We tested this hypothesis with a C₃ and C₄ grass and two caterpillar species with different diet breadths. Lolium multiflorum (C₃) and Bouteloua curtipendula (C₄) were grown in outdoor open top chambers at ambient (370 ppm) or elevated (740 ppm) CO₂. Bioassays compared the performance and digestive efficiencies of Pseudaletia unipuncta (a grass-specialist noctuid) and Spodoptera frugiperda (a generalist noctuid). As expected, the nutritional quality of L. multiflorum changed to a greater extent than did that of B. curtipendula when grown in elevated CO₂; levels of protein (considered growth limiting) declined in the C₃ grass, while levels of carbohydrates (sugar, starch and fructan) increased. However, neither insect species increased its feeding rate on the C₃ grass to compensate for its lower nutritional quality when grown in an elevated CO₂ atmosphere. Consumption rates of P. unipuncta and S. frugiperda were higher on the C₃ grass than the C₄ grass, the opposite of the result expected for a compensatory response to the lower nutritional quality of the C₄ grass. Although our results do not support the hypothesis that grass-specialist insects compensate for lower nutritional quality by increasing their consumption rates more than do generalist insects, the performance of the specialist was greater than that of the generalist on each grass species and at both CO₂ levels. Mechanisms other than compensatory feeding, such as increased nutrient assimilation efficiency, appear to determine the relative performance of these herbivores. Our results also provide further evidence against the hypothesis that C₄ grasses would be avoided by insect herbivores because a large fraction of their nutrients is unavailable to herbivores. Instead, our results are consistent with the hypothesis that C₄ grasses are poorer host plants primarily because of their lower nutrient levels, higher fiber levels, and greater toughness.     

hCG treatment raises H<Subscript>2</Subscript>O<Subscript>2</Subscript> levels and induces germ cell apoptosis in rat testis

The clinical significance of exogenous hCG treatment is to stimulate steroidogenesis and spermatogenesis in the testis. However, the pathogenesis of detrimental effects on the testis arising out of chronic hCG treatment is yet to be clearly ascertained. In the present study we have shown that hCG treatment (100 IU/day) to rats for 30 days raises testicular oxidative stress leading to germ cell apoptosis and impairment of spermatogenesis. The treatment raises testicular H₂O₂ levels along with increase in lipid peroxidation and concomitant decrease in the enzymatic antioxidant activities like superoxide dismutase, catalase and glutathione-s-transferase. The rise in the number of apoptotic germ cells was associated with up regulation of Fas protein expression and caspase-3 activity in the testis. However, serum testosterone which was elevated by 15 days of hCG treatment declined to pretreatment levels by 30 days. No significant alteration in serum gonadotropins was observed. The above findings indicate that the pathogenesis of deleterious effects following chronic hCG treatment is due to increase in testicular oxidative stress with high H₂O₂ availability leading to apoptosis among germ cells.     

Bonding,aromaticity, and planar tetracoordinated carbon in Si<Subscript>2</Subscript>CH<Subscript>2</Subscript> and Ge<Subscript>2</Subscript>CH<Subscript>2</Subscript>

Natural bond orbital (NBO) analyses and dissected nucleus-independent chemical shifts (NICS _{π z z} ) were computed to evaluate the bonding (bond type, electron occupation, hybridization) and aromatic character of the three lowest-lying Si₂CH₂ (1-Si, 2-Si, 3-Si) and Ge₂CH₂ (1-Ge, 2-Ge, 3-Ge) isomers. While their carbon C₃H₂ analogs favor classical alkene, allene, and alkyne type bonding, these Si and Ge derivatives are more polarizable and can favor “highly electron delocalized”? and “non-classical”? structures. The lowest energy Si ₂CH₂ and Ge ₂CH₂ isomers, 1-Si and 1-Ge, exhibit two sets of 3–center 2–electron (3c-2e) bonding; a π-3c-2e bond involving the heavy atoms (C–Si–Si and C–Ge–Ge), and a σ-3c-2e bond (Si–H–Si, Ge–H–Ge). Both 3-Si and 3-Ge exhibit π and σ-3c-2e bonding involving a planar tetracoordinated carbon (ptC) center. Despite their highly electron delocalized nature, all of the Si₂CH₂ and Ge₂CH₂ isomers considered display only modest two π electron aromatic character (NICS(0) _{π z z} =--6.2 to –8.9 ppm, computed at the heavy atom ring center) compared to the cyclic-C ₃H₂ (–13.3 ppm).

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Graphical Abstract The three lowest Si2CH2 and Ge2CH2 isomers.

     

Production of fumonisins B<Subscript>2</Subscript> and B<Subscript>4</Subscript> in <Emphasis Type="Italic">Tolypocladium</Emphasis> species

Tolypocladium inflatum is known primarily for its production of the cyclosporines that are used as an immunosuppressive drug. However, we report here the production of the carcinogenic fumonisins B₂ and B₄ by this biotechnologically relevant fungal genus. These mycotoxins were detected in 11 strains tested from three species: Tolypocladium inflatum, T. cylindrosporum, and T. geodes. Production of fumonisins by Fusarium spp. and Aspergillus niger is highly medium- and temperature-dependent, so the effect of these parameters on fumonisin production by three T. inflatum strains was studied. Maximum production was achieved on media with high sugar content incubated at 25–30°C. Since these results demonstrate that fumonisin production could be widespread within the genus Tolypocladium, the potential contamination of commercial cyclosporine preparations with fumonisins needs to be investigated.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H₂O₂ on cytosolic as well as mitochondrial calcium (Ca²⁺) concentrations, mitochondrial inner membrane potential (_m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H₂O₂, in the absence of extracellular Ca²⁺ ([Ca²⁺]_o) led to an increase either in cytosolic and in mitochondrial Ca²⁺ concentration. Additionally, H₂O₂ induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H₂O₂ on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H₂O₂. However, the H₂O₂-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca²⁺ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H₂O₂-evoked changes in mitochondrial Ca²⁺ concentration but not those in membrane potential and FAD state. The present results have indicated that H₂O₂ can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H₂O₂. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas. (Mol Cell Biochem 269: 165–173, 2005)     

Superoxide dismutase activity in C<Subscript>3</Subscript> and C<Subscript>3</Subscript>/CAM intermediate species of <Emphasis Type="Italic">Clusia</Emphasis>

The C₃-CAM intermediate Clusia minor L. and the C₃ obligate Clusia multiflora H.B.K. plants were exposed for 7 d to a combination of drought stress and high irradiance of about 1200 μmol m⁻² s⁻¹ for 12 h per day. In both species under these conditions a strong decrease in stomatal conductance was observed at dawn and dusk. Changes in stomatal behaviour of C. minor were accompanied by only a low nocturnal accumulation of malate and citrate. Thus, in C. minor drought stress applied in combination with high irradiance limited CAM expression, and possibly this is the main reason why C. minor prefers semi-shaded sites in the field. The mitochondrial MnSOD, in both well watered and stressed plants of two species showed strong diurnal oscillations with maximum activity at dusk. These oscillations can be explained by the engagement of mitochondria in dissipation of an excess of reducing equivalents. In plants which are able to carry out CAM metabolism tricarboxylic acid cycle is expected to be down regulated in the dark period to prevent breakdown of the entire malate and citrate.     

Effects of salinity on chlorophyll fluorescence and CO<Subscript>2</Subscript> fixation in C<Subscript>4</Subscript> estuarine grasses

The effects of salinity (sea water at 0 ‰ versus 30 ‰) on gross rates of O₂ evolution (J _O2) and net rates of CO₂ uptake (P _N) were measured in the halotolerant estuarine C₄ grasses Spartina patens, S. alterniflora, S. densiflora, and Distichlis spicata in controlled growth environments. Under high irradiance, salinity had no significant effect on the intercellular to ambient CO₂ concentration ratio (C _i/C _a). However, during photosynthesis under limiting irradiance, the maximum quantum efficiency of CO₂ fixation decreased under salinity across species, suggesting there is increased leakage of the CO₂ delivered to the bundle sheath cells by the C₄ pump. Growth under salinity did not affect the maximum intrinsic efficiency of photosystem 2, PS2 (F_V/F_M) in these species, suggesting salinity had no effect on photosynthesis by inactivation of PS2 reaction centers. Under saline conditions and high irradiance, P _N was reduced by 75 % in Spartina patens and S. alterniflora, whereas salinity had no effect on P _N in S. densiflora or D. spicata. This inhibition of P _N in S. patens and S. alterniflora was not due to an effect on stomatal conductance since the ratio of C _i/C _a did not decrease under saline conditions. In growth with and without salt, P _N was saturated at ∼500 μmol(quantum) m⁻² s⁻¹ while J _O2 continued to increase up to full sunlight, indicating that carbon assimilation was not tightly coupled to photochemistry in these halophytic species. This increase in alternative electron flow under high irradiance might be an inherent function in these halophytes for dissipating excess energy.     

Intracellular position of mitochondria in mesophyll cells differs between C<Subscript>3</Subscript> and C<Subscript>4</Subscript> grasses

In C₃ plants, part of the CO₂ fixed during photosynthesis in chloroplasts is released from mitochondria during photorespiration by decarboxylation of glycine via glycine decarboxylase (GDC), thereby reducing photosynthetic efficiency. The apparent positioning of most mitochondria in the interior (vacuole side of chloroplasts) of mesophyll cells in C₃ grasses would increase the efficiency of refixation of CO₂ released from mitochondria by ribulose 1,5-bisphosphate carboxylase/?oxygenase (Rubisco) in chloroplasts. Therefore, in mesophyll cells of C₄ grasses, which lack both GDC and Rubisco, the mitochondria ought not to be positioned the same way as in C₃ mesophyll cells. To test this hypothesis, we investigated the intracellular position of mitochondria in mesophyll cells of 14 C₄ grasses of different C₄ subtypes and subfamilies (Chloridoideae, Micrairoideae, and Panicoideae) and a C₃–C₄ intermediate grass, Steinchisma hians, under an electron microscope. In C₄ mesophyll cells, most mitochondria were positioned adjacent to the cell wall, which clearly differs from the positioning in C₃ mesophyll cells. In S. hians mesophyll cells, the positioning was similar to that in C₃ cells. These results suggest that the mitochondrial positioning in C₄ mesophyll cells reflects the absence of both GDC and Rubisco in the mesophyll cells and the high activity of phosphoenolpyruvate carboxylase. In contrast, the relationship between the mitochondrial positioning and enzyme distribution in S. hians is complex, but the positioning may be related to the capture of respiratory CO₂ by Rubisco. Our study provides new possible insight into the physiological role of mitochondrial positioning in photosynthetic cells.     

C<Subscript>4</Subscript> plants in the deserts of China: occurrence of C<Subscript>4</Subscript> photosynthesis and its morphological functional types

C₄ photosynthetic pathway and morphological functional types were determined for 104 species in 45 genera and 10 families from the deserts of China. 67 C₄ species (64.4 %) were found in Dicotyledoneae (e.g. Chenopodiaceae, Polygonaceae, and Amaranthaceae), the other 37 species were in Monocotyledoneae (e.g. Gramineae, Cyperaceae, and Commelinaceae). 36.5 % of the Chenopodiaceae species (predominantly members of the genera Anabasis, Atriplex, Kochia, Salsola, and Suaeda) identified in the desert regions were found with C₄ photosynthesis, which was about 48 % of the total C₄ species. Many C₄ species (58.7 %) were annuals (e.g. Amaranthus, Atriplex, Digitaria, Eragrostis, Kochia, and Salsola) and experienced long-term droughts, high temperature, and high irradiance. Relatively more shrub C₄ species (28 species of 104) were found in Chenopodiaceae (e.g. Anabasis, Camphorosma, Haloxylon, and Salsola) and Polygonaceae (e.g. Calligonum) in the desert regions. Most of shrub C₄ species with small leaf area were no more than 1 m in height and distributed in sandy soils. Composition of relatively more annual species, shrubs, and Chenopodiaceae C₄ species was the primary characteristic for the C₄ species occurrence in deserts, and this was remarkably related with the arid environmental conditions.