Development of an enzymatic procedure to produce high-protein amaranth flour

Summary A basic procedure was developed to produce high-protein amaranth flour (HPAF) using a commercial preparation of heat-stable alpha-amylase. Slurries (20%, w/v) of gelatinized whole flour were liquefied at 70 and 90°C, pH 6.5, 0.1% (w/v) enzyme concentration and 30 min hydrolysis time. Protein content of raw flour was increased from 15 to 29.6 or 39.3% at liquefaction temperatures of 70 or 90°C, respectively. Some physicochemical and functional properties of HPAF were assessed. HPAF might be used as a dry milk extender.     

Mechanism of action of levonorgestrel: in vitro metabolism and specific interactions with steroid receptors in target organs.

Levonorgestrel (LNG) is a synthetic steroid that displays potent progestional and androgenic effects but it lacks estrogen-like activity. To examine the mode of action of this progestin, we studied its metabolism in vitro in target organs and the specific interactions of LNG and its metabolites with putative steroid receptors. The results demonstrated that [³H]LNG was efficiently converted to A-ring reduced derivatives when incubated with rat hypothalamus and pituitary. Under optimal incubation conditions, [³H]5-dihydro LNG (5-LNG) and [³H]3-5-tetrahydro LNG (3,5-LNG) were identified as the major metabolic conversion products, while [³H]3ß, 5-LNG formation occured to a lesser extent. A-ring reduction of LNG was NADPH-dependent. Assessment of the relative binding affinities of LNG and its derivatives to progesterone (PR), androgen (AR) and estrogen (ER) receptors by displacement analysis revealed that unchanged LNG binds with high affinity to PR and AR but not to ER. 5-LNG exhibited a diminished though significant interactions with PR and an enhanced binding affinity for AR as compared with LNG, indicating that 5-reduction of LNG increases its affinity for AR. The most striking finding was that further reduction of the 5-LNG molecule at C-3 abolished its binding activity to PR, AR, and even to ER. The overall data provides a plausible explanation for the lack of estrogen agonistic action of LNG and for its potent progestational and androgenic effects.     

Soil nutrient dissimilarity and litter nutrient limitation as major drivers of home field advantage in riparian tropical forests

Decomposition is a key process driving carbon and nutrient cycling in ecosystems worldwide. The home field advantage effect (HFA) has been found to accelerate decomposition rates when litter originates from “home” when compared to other (“away”) sites. It is still poorly known how HFA plays out in tropical, riparian forests, particularly in forests under restoration. We carried out three independent reciprocal litter transplant experiments to test how litter quality, soil nutrient concentrations, and successional stage (age) influenced HFA in tropical riparian forests. These experimental areas formed a wide gradient of soil and litter nutrients, which we used to evaluate the more general hypothesis that HFA varies with dissimilarity in soil nutrients and litter quality. We found that HFA increased with soil nutrient dissimilarity, suggesting that litter translocation uncouples relationships between decomposers and litter characteristics; and with litter N:P, indicating P limitation in this system. We also found negative HFA effects at a site under restoration that presented low decomposer ability, suggesting that forest restoration does not necessarily recover decomposer communities and nutrient cycling. Within each of the independent experiments, the occurrence of HFA effects was limited and their magnitude was not related to forest age, nor soil and litter quality. Our results imply that HFA effects in tropical ecosystems are influenced by litter nutrient limitation and soil nutrient dissimilarity between home and away sites, but to further disentangle major HFA drivers in tropical areas, a gradient of dissimilarity between litter and soil properties must be implemented in future experimental designs.     

Changes associated with tyrosine phosphorylation during short-term hypoxia in retinal microvascular endothelial cells in vitro

The occlusion of capillary vessels results in low oxygen tension in adjacent tissues which triggers a signaling cascade that culminates in neovascularization. Using bovine retinal capillary endothelial cells (BRCEC), we investigated the effects of short-term hypoxia on DNA synthesis, phosphotyrosine induction, changes in the expression of basic fibroblast growth factor receptor (bFGFR), protein kinase C (PKCα), heat shock protein 70 (HSP70), and SH2-containing protein (SHC). The effect of protein tyrosine kinase (PTK) and phosphatase inhibitors on hypoxia-induced phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO₂ = 20%) conditions were quiesced in low serum containing medium and then exposed to low oxygen tension or hypoxia (pO₂ = 3%) in humidified, 5% CO₂, 37°C, tissue culture chambers, on a time-course of up to 24 h. DNA synthesis was potentiated by hypoxia in a time-dependent manner. This response positively correlated with the cumulative induction of phosphotyrosine and the downregulation of bFGFR (M_r ～ 85 kDa). Protein tyrosine kinase inhibitors, herbimycin-A, and methyl 2,5-dihydroxycinnamate, unlike genistein, markedly blocked hypoxia-induced phosphotyrosine. Prolonged exposure of cells to phosphatase inhibitor, sodium orthovanadate, also blocked hypoxia-induced phosphotyrosine. The expression of HSP70, PKCα, and SHC were not markedly altered by hypoxia. Taken together, these data suggest that short-term hypoxia activates endothelial cell proliferation in part via tyrosine phosphorylation of cellular proteins and changes in the expression of the FGF receptor. Thus, endothelial cell mitogenesis and neovascularization associated with low oxygen tension may be controlled by abrogating signaling pathways mediated by protein tyrosine kinase and phosphatases. © 1995 Wiley-Liss, Inc.     

Neural and endothelial nitric oxide synthase activity in rat penile erectile tissue

NADPH-diaphorase (NADPH-D) activity and immunoreactivity for neural and endothelial nitric oxide synthase (nNOS and eNOS, respectively) were used to investigate nitric oxide (NO) regulation of penile vasculature. Both the histochemical and immunohistochemical techniques for NOS showed that all smooth muscles regions of the penis (dorsal penile artery and vein, deep penile vessels, and cavernosal muscles) were richly innervated. The endothelium of penile arteries, deep dorsal penile vein, and select veins in the crura and shaft were also stained for NADPH-D and eNOS. However, the endothelium of cavernous sinuses was unstained by both techniques. Fewer fibers were seen in the glans penis, those present being associated with small blood vessels and large nerve bundles near the trabecular walls. All penile neurons in the pelvic plexus, located by retrograde transport of a dye placed in the corpora cavernosa penis, were stained by the NADPH-D method. Essentially similar results were obtained with an antibody to nNOS. These data suggest that penile parasympathetic neurons comprise a uniform population, as all seem capable of forming nitric oxide. However, in contrast to the endothelium of penile vessels, the endothelium lining the cavernosal spaces may not be capable of nitric oxide synthesis.     

Long-term treatment with fluoxetine induces desensitization of 5-HT4 receptor-dependent signalling and functionality in rat brain

The mode of action of antidepressant drugs may be related to mechanisms of monoamines receptor adaptation, including serotonin 5-HT₄ receptor subtypes. Here we investigated the effects of repeated treatment with the selective serotonin reuptake inhibitor fluoxetine for 21 days (5 and 10 mg/kg, p.o., once daily) on the sensitivity of 5-HT₄ receptors by using receptor autoradiography, adenylate cyclase assays and extracellular recording techniques in rat brain. Fluoxetine treatment decreased the density of 5-HT₄ receptor binding in the CA1 field of hippocampus as well as in several areas of the striatum over the doses of 5–10 mg/kg. In a similar way, we found a significant lower response to zacopride-stimulated adenylate cyclase activity in the fluoxetine 10 mg/kg/day treated group. Furthermore, post-synaptic 5-HT₄ receptor activity in hippocampus-measured as the excitatory action of zacopride in the pyramidal cells of CA1 evoked by Schaffer collateral stimulation was attenuated in rats treated with both doses of fluoxetine. Taken together, these results support the concept that a net decrease in the signalization pathway of 5-HT₄ receptors occurs after chronic selective serotonin reuptake inhibitor treatment: this effect may underlie the therapeutic efficacy of these drugs.     

Holistic assessment of the microbiome dynamics in the substrates used for commercial champignon (Agaricus bisporus) cultivation

Microorganisms strongly influence and are required to generate the selective substrate that provides nutrients and support for fungal growth, and ultimately to induce mushroom fructification under controlled environmental conditions. In this work, the fungal and bacterial microbiota living in the different substrates employed in a commercial crop (compost phase I, II and III, flush 1 and 2, and casing material on day 1, 6 and 8 after compost casing and during flush 1 and 2) have been characterized along the different stages of cultivation by metataxonomic analysis (16S rRNA and ITS2), analysis of phospholipid fatty acid content (PLFAs) and RT-qPCR. Additionally, laccase activity and the content of lignin and complex carbohydrates in compost and casing have been quantified. The bacterial diversity in compost and casing increased throughout the crop cycle boosted by the connection of both substrates. As reflected by the PLFAs, the total living bacterial biomass appears to be negatively correlated with the mycelium of the crop. Agaricus bisporus was the dominant fungal species in colonized substrates, displacing the pre-eminent Ascomycota, accompanied by a sustained increase in laccase activity, which is considered to be a major product of protein synthesis during the mycelial growth of champignon. From phase II onwards, the metabolic machinery of the fungal crop degrades lignin and carbohydrates in compost, while these components are hardly degraded in casing, which reflects the minor role of the casing for nourishing the crop. The techniques employed in this study provide a holistic and detailed characterization of the changing microbial composition in commercial champignon substrates. The knowledge generated will contribute to improve compost formulations (selection of base materials) and accelerate compost production, for instance, through biotechnological interventions in the form of tailored biostimulants and to design environmentally sustainable bio-based casing materials.     

Ecological specialization and niche overlap of subterranean rodents inferred from DNA metabarcoding diet analysis

Knowledge of how animal species use food resources available in the environment can increase our understanding of many ecological processes. However, obtaining this information using traditional methods is difficult for species feeding on a large variety of food items in highly diverse environments. We amplified the DNA of plants for 306 scat and 40 soil samples, and applied an environmental DNA metabarcoding approach to investigate food preferences, degree of diet specialization and diet overlap of seven herbivore rodent species of the genus Ctenomys distributed in southern and midwestern Brazil. The metabarcoding approach revealed that these species consume more than 60% of the plant families recovered in soil samples, indicating generalist feeding habits of ctenomyids. The family Poaceae was the most common food resource retrieved in scats of all species as well in soil samples. Niche overlap analysis indicated high overlap in the plant families and molecular operational taxonomic units consumed, mainly among the southern species. Interspecific differences in diet composition were influenced, among other factors, by the availability of resources in the environment. In addition, our results provide support for the hypothesis that the allopatric distributions of ctenomyids allow them to exploit the same range of resources when available, possibly because of the absence of interspecific competition.     

Brevipalpus mites associated with coffee plants (Coffea arabica and C. canephora) in Chiapas,Mexico

Experimental and Applied Acarology - Tenuipalpid mites of the genus Brevipalpus are of significant economic and quarantine importance in agriculture. They can damage and vector phytopathogenic...