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.     

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.     

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.     

Molecular dating for phylogenies containing a mix of populations and species by using Bayesian and RelTime approaches

Simultaneous molecular dating of population and species divergences is essential in many biological investigations, including phylogeography, phylodynamics and species delimitation studies. In these investigations, multiple sequence alignments consist of both intra‐ and interspecies samples (mixed samples). As a result, the phylogenetic trees contain interspecies, interpopulation and within‐population divergences. Bayesian relaxed clock methods are often employed in these analyses, but they assume the same tree prior for both inter‐ and intraspecies branching processes and require specification of a clock model for branch rates (independent vs. autocorrelated rates models). We evaluated the impact of a single tree prior on Bayesian divergence time estimates by analysing computer‐simulated data sets. We also examined the effect of the assumption of independence of evolutionary rate variation among branches when the branch rates are autocorrelated. Bayesian approach with coalescent tree priors generally produced excellent molecular dates and highest posterior densities with high coverage probabilities. We also evaluated the performance of a non‐Bayesian method, RelTime, which does not require the specification of a tree prior or a clock model. RelTime's performance was similar to that of the Bayesian approach, suggesting that it is also suitable to analyse data sets containing both populations and species variation when its computational efficiency is needed.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

The role of partial incubation and egg repositioning within the clutch in hatching asynchrony and subsequent effects on breeding success

The main mechanism to achieve hatching asynchrony (HA) for incubating birds is to start heating the eggs before clutch completion. This might be achieved through partial incubation and/or early incubation. Even in the absence of incubation behaviour during the laying phase, clutches still experience a certain degree of asynchrony. Recent studies have shown that eggs located in the centre of the nest receive more heat than peripheral ones during incubation. As eggs receiving more heat would develop faster, we hypothesized that HA should be shorter in nests where eggs were moved homogeneously along the centre–periphery space during incubation than in those nests where eggs repeatedly remained in the same locations, either centrally or peripherally. We explored the relative roles of egg repositioning and partial incubation in determining HA in wild birds by (1) removing eggs from 20 Great Tit Parus major nests on the day of laying and replacing them with fake eggs to avoid partial incubation, and returning them when full incubation began; (2) monitoring twice a day the position of each individually marked egg relative to the clutch centre during incubation, and estimating the coefficient of variation of the distances; and (3) determining HA in each nest. Preventing partial incubation reduced HA by 51% days in experimental nests. It also caused negative effects for the incubating females (lengthening the full incubation period) and positive effects for the brood (increasing fledging success). However, our hypothesis about the role of egg repositioning on HA was not supported: all the females moved the eggs with remarkable consistency, generally attaining a coefficient of variation of the distances around 33%, and it was not related to the HA experienced. We therefore conclude that partial incubation is an important factor regulating HA, and females compensate for the potential effects of differential heating by moving the eggs homogeneously within the clutch.     

Evolution of Cyclic Amidohydrolases: A Highly Diversified Superfamily

Dihydroorotases are universal proteins catalyzing the third step of pyrimidine biosynthesis. These zinc metalloenzymes belong to the superfamily of cyclic amidohydrolases, comprising also other enzymes that are involved in degradation of either purines (allantoinases), pyrimidines (dihydropyrimidinases) or hydantoins (hydantoinases). The evolutionary relationships between these mechanistically related enzymes were estimated after designing a method to build an accurate multiple sequence alignment. The amino acid sequences that have been crystallized were used to build a seed alignment. All the remaining homologues were progressively added by aligning their HMM profiles to the seed HMM profile, allowing to obtain a reliable phylogeny of the superfamily. This helped us to propose a new evolutionary classification of dihydroorotases into three major types, while at the same time disentangling an important part of the history of their complex structure–function relationships. Although differing in their substrate specificity, allantoinases, hydantoinases and dihydropyrimidinases are found to be phylogenetically closer to DHOase Type I than the proximity of the three DHOase types to each other. This suggests that the primordial cyclic amidohydrolase was a multifunctional, highly evolvable generalist, with high conformational diversity allowing for promiscuous activities. Then, successive gene duplications allowed resolving the primordial substrate ambiguity in various substrate specificities. The present-day superfamily of cyclic amidohydrolases is the result of the progressive divergence of these ancestral paralogous copies by descent with modification.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.