Antidepressant-like effect of Cecropia glazioui Sneth and its constituents – In vivo and in vitro characterization of the underlying mechanism

The present study aimed to characterize the antidepressant-like effect of a standardized aqueous extract (AE) of Cecropia glazioui Sneth and its purified fractions on in vivo (forced swimming test), ex vivo (hippocampal monoamines levels) and in vitro (serotonin, noradrenaline and dopamine uptake) tests, searching for the active principles and the underlying mechanisms of action. Treatment with AE, or with its butanolic fraction (BuF), the latter rich in catechins, procyanidins and flavonoids, reduced the immobility of rats in the forced swimming test indicating an antidepressant-like effect. Biochemical analysis of the hippocampal neurotransmitters in BuF-treated rats showed significant increase in monoamines levels. BuF and six of its purified constituents inhibited the uptake of [(3)H]-serotonin, [(3)H]-dopamine and [(3)H]-noradrenaline by synaptosomes of different brain regions. Catechin, catechin (4alpha-->8) ent-catechin (Procyanidin B3 isomer) and epicatechin (4beta-->8) epicatechin (Procyanidin B2) were the most active compounds. Comparatively, the uptake of [(3)H]-noradrenaline was the most affected. These results show that the antidepressant-like effect promoted by C. glazioui extract is most likely due to the blockade of the monoamines uptake in the CNS.     

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy in order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure and conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules (the increased h₊₁/h₀ parameter of maleimide spin label, MSL; 0.377 ± 0.021 in diabetics vs 0.338 ± 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (τ_c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 ± 0.42 in diabetics, n = 21 vs 4.79 ± 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 ± 1.08, n = 28 vs 7.31 ± 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h₊₁/h₀ parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.     

Assessment of a brown midrib (BMR) mutant gene on the nutritive value of sudangrass using in vitro and in vivo techniques

The brown midrib (BMR) gene has been reported to reduce the lignin concentration in plants, which contributed to increased fiber digestion in ruminants. Three studies were completed to compare the digestibility of a BMR mutant of sudangrass (sorghum bicolor subsp. Drummondii) versus a non-BMR (‘Piper’) variety when included in diets fed to sheep (Study 1), to complete a rumen in vitro assessment of sheep and lactating cow diets (Study 2), and to compare digestibility when included in the diet fed to lactating dairy cows (Study 3). Four wether sheep were used in a 2 × 2 Latin square experiment (Study 1) with total fecal collection to determine total tract apparent digestibility of pelleted Piper (P) and BMR (P-BMR) sudangrass hays. Forage pellets consisted of either P-BMR or P hay with added urea to meet the maintenance crude protein (CP) requirement of the sheep. Digestibility of organic matter (OM; P<0.01), dry matter (DM; P<0.01), acid detergent fiber (ADF; P<0.05), and neutral detergent fiber (aNDFom; P<0.07) was higher for P-BMR than P sudangrass. In vitro rumen digestibility of aNDFom using cattle rumen fluid was higher at 24 (P<0.01), 48 (P<0.01) and 72 h (P<0.01) of fermentation for P-BMR versus P (Study 2). Four lactating Holstein dairy cows (251 ± 30 days in milk) and fitted with ruminal and duodenal cannulae were used in a 4 × 4 Latin square experiment. Total mixed rations (TMR) contained 180 g/kg DM shredded sudangrass hay and 180 g/kg sliced alfalfa hay, but the proportion of P to P-BMR sudangrass varied as 100:0, 66:34, 34:66, or 0:100. Yields of milk and milk protein were highest at the 66:34 level (Quadratic: P=0.06 and 0.07, respectively), but composition of milk fat, protein and lactose, as well as DM intake, did not differ (Study 3), probably because forestomach and total tract apparent digestion of aNDFom and OM did not differ due to sudangrass source.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay.Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 μg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 μg/ml for 3 and 20 h in the absence of S9, and for 3 h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 μg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 μg/kg, the last dose being administered 2 h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays.TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Inhibitory effects of indole α‐lipoic acid derivatives on nitric oxide production in LPS/IFNγ activated RAW 264.7 macrophages

Alpha‐lipoic acid (α‐lipoic acid) is a potent antioxidant compound that has been shown to possess anti‐inflammatory effects. RAW 264.7 macrophages produce various inflammatory mediators such as nitric oxide, IL‐1β, IL‐6 and TNF‐alpha upon activation with LPS ( Lipopolysaccharide) and IFNγ (interferon gamma). In this study, the effect of 12 synthetic indole α‐lipoic acid derivatives on nitric oxide production and iNOS (inducible nitric oxide synthase) protein expression in LPS/IFNγ activated RAW 264.7 macrophages was determined. Cell proliferation, nitric oxide levels and iNOS protein expression were examined with thiazolyl blue tetrazolium blue test, griess assay and western blot, respectively. Our results showed that all of the indole α‐lipoic acid derivatives showed significant inhibitory effects on nitric oxide production and iNOS protein levels (p < 0.05). The most active compounds were identified as compound I‐4b, I‐4e and II‐3b. In conclusion, these indole α‐lipoic acid derivatives may have the potential for treatment of inflammatory conditions related with high nitric oxide production. Copyright © 2015 John Wiley & Sons, Ltd.     

Application of Mu in vitro transposition for high-precision mapping of protein–protein interfaces on a yeast two-hybrid platform

High-precision mapping of regions involved in protein–protein interfaces of interacting protein partners is an essential component on a path to understand various cellular functions. Transposon-based systems, particularly those involving in vitro reactions, offer exhaustive insertion mutant libraries and high-throughput platforms for many types of genetic analyses. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision that is based on transposition-assisted construction, sampling, and analysis of a comprehensive insertion mutant library. The methodology integrates random pentapeptide mutagenesis of proteins, yeast two-hybrid screening, and high-resolution genetic footprinting. This straightforward strategy is general, and it provides a rapid and easy means to identify critical contact regions in proteins without the requirement of prior structural knowledge.     

Biological control of brown rot (Moniliana laxa Ehr.) on apricot (Prunus armeniaca L. cv. Hacıhaliloğlu) by Bacillus, Burkholdria, and Pseudomonas application under in vitro and in vivo conditions

In 2002 and 2003, a study was conducted to determine the effect of bacterial strains, Burkholdria OSU 7, Bacillus OSU 142, and Pseudomonas BA 8, on biological control of brown rot disease (Monilinia laxa Ehr.) on apricot cv. Hacıhaliloğlu in Malatya province of Turkey. Apricot orchard at full blooming stage was inoculated with conidial suspension (1 × 10⁶ spores/ml) of M. laxa Ehr. After inoculation, two apricot trees for each application were treated with each of the three biological control agents (Burkholdria gladii OSU 7, Bacillus subtilis OSU 142, and Pseudomonas putida BA 8) by spraying (1 × 10⁹ cfu/ml) on inoculated branches. Disease incidence was evaluated for untreated (control 1) and four different treatment groups including commercial disease management (control 2, positive control: 3% Bourdox in fall, 50% Cupper at pink flower, 30 g/100 l Corus at first blooming, and 300 g/100 l Captan at last blooming stage) and treatments including each of the three bacterial strains (OSU 7, OSU 142, and BA 8). The results showed that disease incidence for negative control (control 1) was 9.94, which was significantly higher than disease incidence for commercial application (2.57%) or bacterial treatments (2.82–5.00%) in the first year. In 2003, the lowest disease incidence observed in OSU 7 treatment (6.80%), while disease incidence rate for positive control and negative control were 9.45% and 28.46%, respectively. This result may suggest that OSU 7 has potential to be used as biopesticide for effective management of brown rot disease on apricot.     

Increases of M2a macrophages and fibrosis in aging muscle are influenced by bone marrow aging and negatively regulated by muscle‐derived nitric oxide

Muscle aging is associated with changes in myeloid cell phenotype that may influence age‐related changes in muscle structure. We tested whether preventing age‐related reductions in muscle neuronal nitric oxide synthase (nNOS) would obviate age‐related changes in myeloid cells in muscle. Our findings show that muscle aging is associated with elevations of anti‐inflammatory M2a macrophages that can increase muscle fibrosis. Expression of a muscle‐specific nNOS transgene in mice prevented age‐related increases in M2a macrophages. Transgene expression also reduced expression of collagens and decreased muscle fibrosis. The nNOS transgene prevented age‐related increases in arginase‐1 but did not influence TGFβ expression, indicating that the transgene may prevent age‐related muscle fibrosis by inhibiting the arginase‐dependent profibrotic pathway. Although aged satellite cells or fibro‐adipogenic precursor (FAPs) cells also promote fibrosis, transgene expression had no effect on the expression of key signaling molecules that regulate fibrogenic activity of those cells. Finally, we tested whether increases in M2a macrophages and the associated increase in fibrosis were attributable to aging of myeloid lineage cells. Young bone marrow cells (BMCs) were transplanted into young or old mice, and muscles were collected 8 months later. Muscles of young mice receiving young BMCs showed no effect on M2a macrophage number or collagen accumulation compared to age‐matched, nontransplanted controls. However, muscles of old mice receiving young BMCs showed fewer M2a macrophages and less accumulation of collagen. Thus, the age‐related increase in M2a macrophages in aging muscle and the associated muscle fibrosis are determined in part by the age of bone marrow cells.     

A >22 Å long metal-complex anion and a 1D cationic coordination polymer, both based on 4,4′-bipyridine-N,N′-dioxide, yielding a 3D and 2-fold interpenetrated fsc net of the hydrogen-bonded metal-organic 1D polymer

The cationic one-dimensional (1D) coordination polymer chain ¹_∞{[Co(μ-bpdo)(H₂O)₄]²⁺} and the metal-complex anion trans-[Co(SO₄)₂(bpdo)₂(H₂O)₂]²⁻, both based on the 4,4′-bipyridine-N,N′-dioxide (bpdo) ligand, form a complementary supramolecular pair ¹_∞{[Co(μ-bpdo)(H₂O)₄]²⁺}_nn[Co(SO₄)₂(bpdo)₂(H₂O)₂]²⁻ (1) with respect to charge balance and hydrogen bonding. With a length of >22.14 ? along the bpdo-Co-bpdo axis the metal-complex trans-[Co(SO₄)₂(bpdo)₂(H₂O)₂]²⁻ is one of the longest and anisotropic counter anions (aspect ratio 22.14:8.11:4.17) observed so far in coordination polymers. Hydrogen-bonding of the anion links the cationic metal-organic 1D polymer into a 2-fold interpenetrated three-dimensional (3D) fsc (or sqc11) 4,6-c 2-nodal net of stoichiometry (4-c)(6-c) with square-planar, 4-connected (Co in anion) and octahedral, 6-connected (Co in cation) nodes in a 1:1 ratio. The 4-c point symbol is (4⁴.6²), the 6-c one (4⁴.6¹⁰.8) yielding a point symbol for the fsc net of (4⁴.6²)(4⁴.6¹⁰.8). The synthesis of 1 requires the presence of a Schiff base. Synthesis under the same conditions in the absence of the Schiff base yields the molecular complex and cocrystal [Co(bpdo)(H₂O)₅]SO₄·1/2bpdo (2) which is related (as pseudo-polymorph) to the known solvate [Co(bpdo)(H₂O)₅]SO₄·2H₂O (3) (CSD Refcodes RAXMUZ and RAXMUZ01).     

Land-use history and succession of Larix decidua in the Southern Alps of Italy–An essay based on a cultural history study of Roswitha Asche

Larix decidua, the European Larch tree, is a typical forest species of the Southern Alps of North Italy growing in mixture with Picea, in isolated pure stands, or as solitary trees on meadows at 1000–2000 m elevation. Presently, the old-growth Larix forests show no regeneration, while Picea abies, Norway spruce, invades at lower elevation, and Pinus cembra, Stone pine, at higher elevation. In this study, we investigate the conditions that lead to pure stands of L. decidua in the past. It is shown that intensive land-use of grazing was essential for Larix regeneration, and that the pure stands of Larix will disappear in the future with the change of land-use, unless management will be intensified again to produce mineral soil for Larix to regenerate.     

Epidemic Development and Virulence in 1995–1998 of Puccinia coronata, a Potential Biocontrol Agent of Wild Oats on San Clemente Island

The biology of islands differs from that of large land masses in having less complex ecosystems. Introduced exotic weeds are often a major threat to fragile island ecosystems because of their expansion into habitats previously occupied by endemic species. San Clemente Island, 120 km off the California coastline, is an example of this process in which numerous exotic weed species have been introduced and some are endangering the native flora. Crown rust of oats caused by Puccinia coronata f.sp. avenae was investigated as a potential biocontrol agent against two wild oat species, Avena barbata and Avena fatua, introduced on San Clemente Island. Epidemiology and virulence of this rust were studied. The island was surveyed from 1995 to 1998 for occurrence of P. coronata on wild oats. Wild oats were found sprouting in the northern part of the island shortly after autumn rainfall and subsequently covered the main grasslands of the island. The rust also appeared first on the northern part of the island and progressively spread south. However, disease severities in the south were considerably lower than those in the north. Diverse virulence types, although related to Californian and Mexican forms, were detected among the isolates. The potential use of P. coronata as an augmentative biocontrol agent for wild oat species on San Clemente Island is discussed.