A red-emitting carborhodamine for monitoring and measuring membrane potential.

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.

A high-affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs.

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 µM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert-Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype ("L-type") of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP's mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1-10 µM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1-1 mM) antagonist activity. We also showed that 1.5-µM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-µM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-µM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.

The Frog Motor Nerve Terminal Has Very Brief Action Potentials and Three Electrical Regions Predicted to Differentially Control Transmitter Release.

The action potential (AP) waveform controls the opening of voltage-gated calcium channels and contributes to the driving force for calcium ion flux that triggers neurotransmission at presynaptic nerve terminals. Although the frog neuromuscular junction (NMJ) has long been a model synapse for the study of neurotransmission, its presynaptic AP waveform has never been directly studied, and thus the AP waveform shape and propagation through this long presynaptic nerve terminal are unknown. Using a fast voltage-sensitive dye, we have imaged the AP waveform from the presynaptic terminal of male and female frog NMJs and shown that the AP is very brief in duration and actively propagated along the entire length of the terminal. Furthermore, based on measured AP waveforms at different regions along the length of the nerve terminal, we show that the terminal is divided into three distinct electrical regions: A beginning region immediately after the last node of Ranvier where the AP is broadest, a middle region with a relatively consistent AP duration, and an end region near the tip of nerve terminal branches where the AP is briefer. We hypothesize that these measured changes in the AP waveform along the length of the motor nerve terminal may explain the proximal-distal gradient in transmitter release previously reported at the frog NMJ.SIGNIFICANCE STATEMENT The AP waveform plays an essential role in determining the behavior of neurotransmission at the presynaptic terminal. Although the frog NMJ is a model synapse for the study of synaptic transmission, there are many unknowns centered around the shape and propagation of its presynaptic AP waveform. Here, we demonstrate that the presynaptic terminal of the frog NMJ has a very brief AP waveform and that the motor nerve terminal contains three distinct electrical regions. We propose that the changes in the AP waveform as it propagates along the terminal can explain the proximal-distal gradient in transmitter release seen in electrophysiological studies.

Probiotic Potential of Lactobacillus paracasei CT12 Isolated from Water Kefir Grains (Tibicos).

The water kefir grains are a multi-species starter culture used to produce fermented beverages of sucrose solution with or without fruit extracts. The water kefir grains are known in Mexico as Tibicos, which are mainly used to produce Tepache, a traditional Mexican drink made by fermenting pineapple peel. The microbiota of Tibicos mainly include lactic acid bacteria (LAB) and since most probiotics belong to this group, Tibicos may represent a potential source of probiotic bacteria. Moreover, several bacteria isolated from kefir samples have been recognized as probiotics. Hence, the aim of this study was to assess the probiotic properties of a Lactobacillus strain isolated from Tibicos. The isolated, designed as CT12, was identified as Lactobacillus paracasei by sequencing 16S RNA gene. L. paracasei CT12 showed a survival rate of ca. 57% and 40% following simulated gastric and intestinal digestion, respectively. Besides, the strain was sensitive to ampicillin and erythromycin, and exhibited hydrophobicity (97-99%), autoaggregation (ca. 70%) and mucin adhesion properties (up to 90%), while no possessed haemolytic capacity. Furthermore, its cell-free supernatant displayed relevant antimicrobial, antifungal and antioxidant capacity. Hence, L. paracasei CT12 appears to possess a potential probiotic value.

Retrograde enteroclysis by double balloon enteroscopy in a patient with blunt abdominal trauma: small bowel stricture, intraluminal vascular lesion and Crohn's disease.

A 40-year-old male presented to the Emergency Department after a driving accident with blunt abdominal trauma. An abdominal computed tomography (CT) scan revealed a mesenteric injury in the right lower quadrant. He was admitted two months later due to a one-day history of abdominal pain and diarrhea, without fever or blood. The CT angiography showed a pseudoaneurysm located in the proximal ileum and several rigid small bowel (SB) loops with segmental wall thickening of mucosa.

Synthesis of Sulfonated Carbofluoresceins for Voltage Imaging.

We present the design, synthesis, and applications of a new class of voltage-sensitive fluorescent indicators built on a modified carbofluorescein scaffold. Carbofluoresceins are an attractive target for responsive probes because they maintain oxygen substitution patterns at the 3' and 6' positions, similar to fluorescein, while simultaneously possessing excitation and emission profiles red-shifted nearly 50 nm compared to fluorescein. However, the high p Ka of carbofluorescein dyes, coupled with their tendency to cyclize to nonfluorescent configurations, precludes their use in voltage-imaging applications. Here, we overcome the limitations of carbofluoresceins via chlorination to lower the p Ka by 2 units to 5.2 and sulfonation to prevent cyclization to the nonabsorbing form. To achieve this, we devise a synthetic route to halogenated sulfonated carbofluoresceins from readily available, inexpensive starting materials. New, chlorinated sulfone carbofluoresceins have low p Ka values (5.2) and can be incorporated into phenylenevinylene molecular wire scaffolds to create carboVoltage-sensitive fluorophores (carboVF dyes). The best of the new carboVF dyes, carboVF2.1(OMe).Cl, possesses excitation and emission profiles of >560 nm, displays high voltage sensitivity (>30% Δ F/ F per 100 mV), and can be used in the presence of other blue-excited fluorophores such as green fluorescent protein. Because carboVF2.1(OMe).Cl contains a phenolic oxygen, it can be incorporated into fluorogenic labeling strategies. Alkylation with a sterically bulky cyclopropylmethyl-derived acetoxymethyl ether renders carboVF weakly fluorescent; we show that fluorescence can be restored by the action of porcine liver esterase both in vitro and on the surface of living cells and neurons. Together, these results suggest chlorinated sulfone carbofluoresceins can be promising candidates for hybrid chemical-genetic voltage imaging at wavelengths beyond typical fluorescein excitation and emission.

Protective Effect of Chickpea Protein Hydrolysates on Colon Carcinogenesis Associated With a Hypercaloric Diet.

OBJECTIVE: Colon cancer occupies the third place in incidence worldwide; eating habits, in particular, consumption of hypercaloric diets, are relevant in its etiopathogenesis. On the other hand, foods can also modulate carcinogenesis: for example, proteins, which when hydrolyzed release peptides with biological activities, and legumes, especially, chickpea, represent a good source of hydrolysates. The objective of this work was to verify the inhibitory effect of chickpea hydrolyzed protein on azoxymethane (AOM)-induced carcinogenesis in mice fed a hypercaloric diet. METHODS: We hydrolyzed chickpea protein by pepsin, pancreatin, and a combined pepsin-pancreatin system, to test its anticarcinogenic and hypercaloric activity in mice that had consumed a hypercaloric diet or a normal diet but were injected with azoxymethane (AOM). RESULTS: A concentrate (70% proteins) was obtained from chickpea seeds (18.5% proteins), and extensive hydrolysates were obtained at 15 minutes, in all tested enzyme systems. The greatest activity was evidenced in the hydrolysates obtained with pepsin-pancreatin at 90 minutes. Animals that consumed the hypercaloric diet had a higher concentration of cholesterol and a higher atherogenic index, which were significantly reduced with the administration of chickpea protein hydrolysates with a dose-response effect (10, 20, or 30 mg/kg), whereas no effect was observed in animals that consumed the normal diet. In animals given AOM, aberrant crypts were observed, at a higher rate in animals that consumed the hypercaloric diet; with the consumption of hydrolysates by the animals that consumed either diet, the number of aberrant crypts was reduced with the 3 doses tested, and the effect was better in those animals fed the hypercaloric diet. The best effect in all tests was with 30 mg/kg body weight. CONCLUSION: The consumption of chickpea protein hydrolysates might confer a protective effect against colon carcinogenesis.

Cooked Chickpea Consumption Inhibits Colon Carcinogenesis in Mice Induced with Azoxymethane and Dextran Sulfate Sodium.

SCOPE: The objective of the study was to evaluate the effect of daily consumption of cooked chickpea (2% and 10%) in ICR male mice in which colon cancer was induced with azoxymethane and dextran sulfate sodium. METHODS AND RESULTS: The effect of consumption of 2% or 10% cooked chickpeas on carcinogenesis-induced colon azoxymethane (AOM)/dextran sulfate sodium (DSS) in ICR mice was determined. Protein oxidation and lipids were determined by colorimetric methods and oxidation of DNA through the identification of adducts 8-hydroxy-2'-desoxiguanosine and proliferation markers (proliferating cell nuclear antigen [PCNA], Ki-67, and ß-catenin), and inflammation (cyclooxygenase [COX]-2 and inducible nitric oxide synthase [iNOS]) were identified by immunohistochemistry reactions. The results showed the protective effect of daily consumption of rich cooked chickpeas in the carcinogenesis process, decreasing lipid, protein, and DNA oxidation and decreasing the expression of inflammatory enzymes (COX-2 and iNOS) as well as ß-catenin, one of the most important oncogenic proteins in colon cancer. Animals that were fed with the 10% chickpea diet showed an inhibition in cellular proliferation (Ki-67 and PCNA expression). CONCLUSIONS: The addition of cooked chickpea seed (2% and 10%) to the daily diet is proposed as a chemopreventive agent against colon cancer.

Chronic Multiorgan Rare Disease: The Role of the Nurse Practitioner as a Leader of the Healthcare Team.

Value in healthcare must focus on accessibility, quality, and affordability. This article describes how a healthcare team provides value by meeting the needs of a rare disease patient and underscores the importance of a chronic multiorgan rare disease home. A nurse practitioner can ensure that barriers to evaluation are removed and communication is prioritized in order to provide accessible and affordable care to a patient with rare disease without jeopardizing quality of care.

Virginia Orange: A Versatile, Red-Shifted Fluorescein Scaffold for Single- and Dual-Input Fluorogenic Probes.

Fluorogenic molecules are important tools for biological and biochemical research. The majority of fluorogenic compounds have a simple input-output relationship, where a single chemical input yields a fluorescent output. Development of new systems where multiple inputs converge to yield an optical signal could refine and extend fluorogenic compounds by allowing greater spatiotemporal control over the fluorescent signal. Here, we introduce a new red-shifted fluorescein derivative, Virginia Orange, as an exceptional scaffold for single- and dual-input fluorogenic molecules. Unlike fluorescein, installation of a single masking group on Virginia Orange is sufficient to fully suppress fluorescence, allowing preparation of fluorogenic enzyme substrates with rapid, single-hit kinetics. Virginia Orange can also be masked with two independent moieties; both of these masking groups must be removed to induce fluorescence. This allows facile construction of multi-input fluorogenic probes for sophisticated sensing regimes and genetic targeting of latent fluorophores to specific cellular populations.

Hypocholesterolemic and Anticarcinogenic Effect of Vicia faba Protein Hydrolyzates.

In recent years, the consumption of vegetal-source proteins has been studied to determine their preventing effect on the development of several chronic diseases. The initial purpose of this report was to determine the effect of a hypercholesterolemic diet (HCD) given to mice, alone or with azoxymethane (AOM), on various obesity biochemical biomarkers, as well as on the induction of colon aberrant crypts (aberrant crypt foci; ACF). At the end of the 5-week assay, animals fed the HCD showed alterations in the level of total cholesterol, high- and low-density lipoproteins, and in the Atherogenic Index; besides, a significant elevation was observed in the number of ACF. Our second aim was to examine the effect of a Faba Protein Hydrolyzate (FPH) on mice fed the HCD. We first obtained protein hydrolyzates from the seeds of Vicia faba, determined the in vitro antioxidant potential with two tests, and, subsequently, evaluated the effect on obesity biomarkers and on the number of ACF. In the first case, we found that, generally, the best protective effect was obtained with the low dose of FPH (10 mg/kg) administered to animals fed the HCD, and injected AOM. With respect to the number of ACF, we observed that this dose was more effective, inhibiting such lesions to almost the level determined for the normocholesterolemic diet (NCD). Therefore, our results demonstrated the relevance of a HCD to develop anomalies in obesity biomarkers in mouse, as well as to increase the number of precarcinogenic lesions. Our results also showed a protective response with the administration of FPH, particularly with a specific dose, suggesting the need for extending research on the matter by widening the spectra of doses, in order to clearly define its potential to counteract the damage induced by the HCD, as well as to confirm if antioxidation in mice was involved in such an effect.

Nutrient and nonnutrient components of legumes, and its chemopreventive activity: a review.

Legumes in combination with other products are the staple food for a large part of the world population, especially the low-income fragment, because their seeds provide valuable amounts of carbohydrates, fiber, and proteins, and have an important composition of essential amino acids, the sulphured amino acids being the limiting ones. Furthermore, legumes also have nonnutritional compounds that may decrease the absorption of nutrients or produce toxic effects; however, it has been reported that depending on the dose, these nonnutritional compounds also have different bioactivities as antioxidant, hypolipidemic, hypoglycemic, and anticarcinogenic agents, which have been proven in scientific studies. It has been observed that in countries with a high consumption of legumes, the incidence of colorectal cancer is lower. Some studies have shown that legume seeds are an alternative chemopreventive therapy against various cancers especially colon; this was verified in various animal models of induced by azoxymethane, a colon specific carcinogenic compound, in which a diet was supplemented with different concentrations of beans, lentils, chickpeas, or soybeans, mostly. These studies have proven the anticancer activity of legumes in early stages of carcinogenesis. Therefore, it is important to review the information available to elucidate the chemopreventive mechanisms of action of legume compounds.

DFT 1H-1H coupling constants in the conformational analysis and stereoisomeric differentiation of 6-heptenyl-2H-pyran-2-ones: configurational reassignment of synargentolide A.

Density functional theory (DFT) (1) H-(1) H NMR coupling constant calculations, including solvation parameters with the polarizable continuum model B3LYP/DGDZVP basis set together with the experimental values measured by spectral simulation, were used to predict the configuration of hydroxylated 6-heptenyl-5,6-dihydro-2H-pyran-2-ones 1, 2, 4, and 7, allowing epimer differentiation. Modeling of these flexible compounds requires the inclusion of solvation models that account for stabilizing interactions derived from intramolecular and intermolecular hydrogen bonds, in contrast with peracetylated derivatives (3, 5, and 6) in which the solvation consideration can be omitted. Using this DFT NMR integrated approach as well as spectral simulation, the configurational reassignment of synargentolide A (8) was accomplished by calculations in the gas phase among four possible diastereoisomers (8-11). Calculated (3) JH,H values established its configuration as 6R-[4'S,5'S,6'S-(triacetyloxy)-2E-heptenyl]-5,6-dihydro-2H-pyran-2-one (8), in contrast with the incorrect 6R,4'R,5'R,6'R-diastereoisomer previously proposed by synthesis (12). Application of this approach increases the probability for successful enantiospecific total syntheses of flexible compounds with multiple chiral centers.

Jatropha curcas Protein Concentrate Stimulates Insulin Signaling, Lipogenesis, Protein Synthesis and the PKCα Pathway in Rat Liver.

Jatropha curcas is an oil seed plant that belongs to the Euphorbiaceae family. Nontoxic genotypes have been reported in Mexico. The purpose of the present work was to evaluate the effect of a Mexican variety of J. curcas protein concentrate (JCP) on weight gain, biochemical parameters, and the expression of genes and proteins involved in insulin signaling, lipogenesis, cholesterol and protein synthesis in rats. The results demonstrated that short-term consumption of JCP increased serum glucose, insulin, triglycerides and cholesterol levels as well as the expression of transcription factors involved in lipogenesis and cholesterol synthesis (SREBP-1 and LXRα). Moreover, there was an increase in insulin signaling mediated by Akt phosphorylation and mTOR. JCP also increased PKCα protein abundance and the activation of downstream signaling pathway targets such as the AP1 and NF-κB transcription factors typically activated by phorbol esters. These results suggested that phorbol esters are present in JCP, and that they could be involved in the activation of PKC which may be responsible for the high insulin secretion and consequently the activation of insulin-dependent pathways. Our data suggest that this Mexican Jatropha variety contains toxic compounds that produce negative metabolic effects which require caution when using in the applications of Jatropha-based products in medicine and nutrition.

Knowledge and behavioral effects in cardiovascular health: Community Health Worker Health Disparities Initiative, 2007-2010.

INTRODUCTION: Cardiovascular disease is the leading cause of death in the United States, and disparities in cardiovascular health exist among African Americans, American Indians, Hispanics, and Filipinos. The Community Health Worker Health Disparities Initiative of the National Heart, Lung, and Blood Institute (NHLBI) includes culturally tailored curricula taught by community health workers (CHWs) to improve knowledge and heart-healthy behaviors in these racial/ethnic groups. METHODS: We used data from 1,004 community participants in a 10-session curriculum taught by CHWs at 15 sites to evaluate the NHLBI's health disparities initiative by using a 1-group pretest-posttest design. The curriculum addressed identification and management of cardiovascular disease risk factors. We used linear mixed effects and generalized linear mixed effects models to examine results. RESULTS: Average participant age was 48; 75% were female, 50% were Hispanic, 35% were African American, 8% were Filipino, and 7% were American Indian. Twenty-three percent reported a history of diabetes, and 37% reported a family history of heart disease. Correct pretest to posttest knowledge scores increased from 48% to 74% for heart healthy knowledge. The percentage of participants at the action or maintenance stage of behavior change increased from 41% to 85%. CONCLUSION: Using the CHW model to implement community education with culturally tailored curricula may improve heart health knowledge and behaviors among minorities. Further studies should examine the influence of such programs on clinical risk factors for cardiovascular disease.

Identifying ligands for the PHD1 finger of KDM5A through high-throughput screening.

PHD fingers are a type of chromatin reader that primarily recognize chromatin as a function of lysine methylation state. Dysregulated PHD fingers are implicated in various human diseases, including acute myeloid leukemia. Targeting PHD fingers with small molecules is considered challenging as their histone tail binding pockets are often shallow and surface-exposed. The KDM5A PHD1 finger regulates the catalytic activity of KDM5A, an epigenetic enzyme often misregulated in cancers. To identify ligands that disrupt the PHD1-histone peptide interaction, we conducted a high-throughput screen and validated hits by orthogonal methods. We further elucidated structure-activity relationships in two classes of compounds to identify features important for binding. Our investigation offers a starting point for further optimization of small molecule PHD1 ligands.

Microstructural Changes in Vanilla planifolia Beans after Using High-Hydrostatic-Pressure Treatment in the Curing Process.

During vanilla bean curing, the cell arrangement derived from the killing technique applied to start bean ripening is essential to obtain the characteristic aroma and flavor of vanilla. Hence, killing is an important step to release the enzymes and compounds required for vanillin production. In this work, high hydrostatic pressure (HHP) at 100-400 MPa for 5 min, using water at 7 °C as the pressure-transmitting medium, was applied as the killing method, and its effect on the microstructural changes in vanilla beans during different curing cycles (C0-C20) was evaluated and compared with that observed after scalding by using water at 100 °C for 8 s. Microstructural changes in the cross-sectioned beans were analyzed using a stereomicroscope (SM), confocal laser scanning microscopy (CLSM), and environmental scanning electron microscopy (ESEM). The vanilla beans were cross-sectioned and three main sectors were analyzed: the total, annular, and core. The morphometric descriptors, namely, area, Feret's diameter, and circularity, were quantified via digital image analysis (DIA), from which a shrinkage ratio was calculated. The results show that the total area in the beans presented a maximum decrease in the C16 of curing. The core area was most affected by the HHP treatment, mainly at 400 MPa, rather than scalding. CSLM observations revealed the autofluorescence of the compounds inside the beans. In conclusion, the use of microscopy techniques and DIA allowed us to determine the microstructural changes in the HHP-treated pods, which were found to be more numerous than those found in the scalded beans.

Effect of cooking on structural changes in the common black bean (Phaseolus vulgaris var. Jamapa).

The cooking process is fundamental for bean consumption and to increase the bioavailability of its nutritional components. The study aimed to determine the effect of cooking on bean seed coat through morphological analyses with different microscopy techniques and image analyses. The chemical composition and physical properties of raw black bean (RBB) and cooked black bean (CBB) seeds were determined. The surface and cross-sectional samples were studied by Optical microscopy (OM), environmental scanning electron microscopy (ESEM), atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). The composition of samples showed significant differences after the cooking process. OM images and gray level co-occurrence matrix algorithm (GLCM) analysis indicated that cuticle-deposited minerals significantly influence texture parameters. Seed coat surface ESEM images showed cluster cracking. Texture fractal dimension and lacunarity parameters were effective in quantitatively assessing cracks on CBB. AFM results showed arithmetic average roughness (Ra) (121.67 nm) and quadratic average roughness (Rq) (149.94 nm). The cross-sectional ESEM images showed a decrease in seed coat thickness. The CLSM results showed an increased availability of lipids along the different multilayer tissues in CBB. The results generated from this research work offer a valuable potential to carry out a strict control of bean seed cooking at industrial level, since the structural changes and biochemical components (cell wall, lipids and protein bodies) that occur in the different tissues of the seed are able to migrate from the inside to the outside through the cracks generated in the multilayer structure that are evidenced by the microscopic techniques used.

Effect of post-harvest storage on the chemical and microstructural characteristics of the common bean (Phaseolus vulgaris L.).

Hard to cook is a textural defect that affects the nutritional quality of beans stored under adverse temperature and humidity conditions. This defect is related to intrinsic characteristics such as seed coat thickness, composition and microstructure. The aim of the present study was to evaluate the chemical and microstructural characteristics of common bean (Phaseolus vulgaris L.) during 270 days of post-harvest storage at 30 °C and 70% relative humidity. Microstructural analysis revealed alteration of the cotyledon cell wall and seed coat affecting seed viability and restricting seedling emergence. The seed coat thickness contraction from 105.79 µm to 97.35 µm (270 days). Changes are related with the protein bodies migration from cotyledons to seed coat. An increase in neutral detergent fiber and the presence of CaOx crystals were observed, which confer rigidity to the seed coat and affect water diffusion after 150 days causing permeability changes that contributed to seed hardening.

Angiotensin-converting enzyme-inhibitory activity in protein hydrolysates from normal and anthracnose disease-damaged Phaseolus vulgaris seeds.

BACKGROUND: Bean seeds are an inexpensive source of protein. Anthracnose disease caused by the fungus Colletotrichum lindemuthianum results in serious losses in common bean (Phaseolus vulgaris L.) crops worldwide, affecting any above-ground plant part, and protein dysfunction, inducing the synthesis of proteins that allow plants to improve their stress tolerance. The aim of this study was to evaluate the use of beans damaged by anthracnose disease as a source of peptides with angiotensin-converting enzyme (ACE-I)-inhibitory activity. RESULTS: Protein concentrates from beans spoiled by anthracnose disease and from regular beans as controls were prepared by alkaline extraction and precipitation at isolelectric pH and hydrolysed using Alcalase 2.4 L. The hydrolysates from spoiled beans had ACE-I-inhibitory activity (IC(50) 0.0191 mg protein mL(-1)) and were very similar to those from control beans in terms of ACE-I inhibition, peptide electrophoretic profile and kinetics of hydrolysis. Thus preparation of hydrolysates using beans affected by anthracnose disease would allow for revalorisation of this otherwise wasted product. CONCLUSION: The present results suggest the use of spoiled bean seeds, e.g. anthracnose-damaged beans, as an alternative for the isolation of ACE-I-inhibitory peptides to be further introduced as active ingredients in functional foods.