Simulated gastrointestinal digestion/Caco-2 cell model to predict bioaccessibility and intestinal permeability of p-coumaric acid and p-coumaroyl derivatives in peanut.

Data concerning physiological recovery of whole peanut major phenolics throughout the gastrointestinal tract are scarce. In our study, the bioaccessibility and intestinal permeability of peanuts major phenolics were predicted by simulated digestion followed by Caco-2 cells monolayer model. Phenolics identification and quantification were performed by HPLC-ESI-QTOF-MS and HPLC-PDA, respectively. As results, p-coumaroyl conjugates with tartaric, sinapic and ferulic acids, and p-coumaric acid were the major phenolics found in the non-digested extract and in the digested and transported fractions. The in vitro bioaccessibility and Caco-2 cell transport of p-coumaric acid was 370% and 127%, respectively, while it was much lower for p-coumaroyl derivatives (7-100% and 14-31%, respectively). Nonetheless, the peroxyl scavenging activity remained unaltered, likely, at least partly, due to synergies between some phenolics, which concentration proportions changed throughout the experiment. Hence, there is indication that whole peanut is a source of bioavailable antioxidant phenolics.

Elite Bradyrhizobium strains boost biological nitrogen fixation and peanut yield in tropical drylands.

Peanut (Arachis hypogaea L.) is an important crop for the family-based systems in the tropics, mainly in Brazil. In the Brazilian drylands, peanuts are cropped in low technological systems, and cheap and efficient technologies are needed to improve crop yield and sustainability. Despite this importance, few data are available on selecting efficient peanut rhizobia in experiments under different edaphoclimatic conditions. This work evaluated the agronomic efficiency and the biological nitrogen fixation (BNF) by two elite Bradyrhizobium strains under four different fields in the Brazilian semiarid region. We compared a new efficient strain Bradyrhizobium sp. ESA 123 with the reference strain B. elkanii SEMIA 6144, currently used in peanut rhizobial inoculants in Brazil. Besides the inoculated treatments, two uninoculated controls were assessed (with and without 80 kg ha-1 of N-urea). The BNF was estimated by the δ15N approach in three out of four field assays. BNF contribution was improved by inoculation of both Bradyrhizobium strains, ranging from 42 to 51% in Petrolina and 43 to 60% in Nossa Senhora da Glória. Peanuts' yields benefited from the inoculation of both strains and N fertilization in all four assays. Nevertheless, the results showed the efficiency of both strains under different edaphoclimatic conditions, indicating the native strain ESA 123 as a potential bacterium for recommendation as inoculants for peanuts in Brazil, mainly in drylands.

Optimizing Procedures for Antioxidant Phenolics Extraction from Skin and Kernel of Peanuts with Contrasting Levels of Drought Tolerance.

Peanut is an affordable legume known for its nutritional value and phenolic content. The kernel and skin of 14 peanut genotypes contrasting in drought tolerance had their phenolic profiles determined and reactive oxygen species (ROS) scavenging activity evaluated. Firstly, temperature and % EtOH to extract antioxidant phenolic compounds were optimized using response surface methodology (RSM). The optimized extraction conditions, 60 °C and 35% EtOH for kernels and 40 °C and 60% EtOH for skins, were further adopted, and phenolic compounds were identified and quantified using high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (HPLC-ESI-QTOF-MS) and high-performance liquid chromatography with photodiode array detector (HPLC-PDA). As a result, phenolic acids and glycosidic/non-glycosidic flavonoids were found. Principal component analysis was conducted, and the pairwise score plot of the skin extracts based on individual phenolic compounds showed a trend of genotype clustering based not only on drought tolerance but also on botanical type of germplasm. Therefore, our results demonstrate the status quo for antioxidant phenolic compounds of peanut genotypes contrasting in drought tolerance grown under natural field conditions.

Polyphenol analysis using high-resolution mass spectrometry allows differentiation of drought tolerant peanut genotypes.

BACKGROUND: Peanut (Arachis hypogaea L.) is an important economic food crop highly appreciated worldwide. Although peanut cultivation has been expanding to dry regions, with water stress during growth stages, current genotypes are not adapted to drought. This study aimed to identify and quantify the full range of polyphenols in five peanut genotypes with different degrees of drought tolerance developed by Embrapa (Brazil) using ultra-high-definition accurate-mass liquid chromatography-electrospray ionization-LTQ-Orbitrap-mass spectrometry. RESULTS: Fifty-eight polyphenols of the classes hydroxybenzoic acids, hydroxycinnamic acids, flavanols, flavonols and flavanones were identified and quantified using high mass accuracy data and confirmed by tandem mass spectrometric experiments. High-definition mass spectrometric analyses revealed nine phenolic compounds that have never been reported in peanuts before. Polyphenol assessment using multivariate analysis allowed identification of the level of similarities among the five peanut genotypes studied. CONCLUSION: Higher drought-tolerant genotypes exhibited higher content of flavonoids, which suggests a relationship between these compounds and drought tolerance traits. © 2019 Society of Chemical Industry.

Introgression of wild alleles into the tetraploid peanut crop to improve water use efficiency, earliness and yield.

The introduction of genes from wild species is a practice little adopted by breeders for the improvement of commercial crops, although it represents an excellent opportunity to enrich the genetic basis and create new cultivars. In peanut, this practice is being increasingly adopted. In this study we present results of introgression of wild alleles from the wild species Arachis duranensis and A. batizocoi improving photosynthetic traits and yield in a set of lines derived from the cross of an induced allotetraploid and cultivated peanut with selection under water stress. The assays were carried out in greenhouse and field focusing on physiological and agronomic traits. A multivariate model (UPGMA) was adopted in order to classify drought tolerant lines. Several lines showed improved levels of tolerance, with values similar to or greater than the tolerant control. Two BC1F6 lines (53 P4 and 96 P9) were highlighted for good drought-related traits, earliness and pod yield, having better phenotypic profile to the drought tolerant elite commercial cultivar BR1. These lines are good candidates for the creation of peanut cultivars suitable for production in semiarid environments.

Validating a probe from GhSERK1 gene for selection of cotton genotypes with somatic embryogenic capacity.

Substantial progress is being reported in the techniques for plant transformation, but successful regeneration of some genotypes remains a challenging step in the attempts to transform some recalcitrant species. GhSERK1 gene is involved on embryo formation, and its overexpression enhances the embryogenic competence. In this study we validate a short GhSERK1 probe in order to identify embryogenic cotton genotypes using RT-qPCR and blotting assays. Cotton genotypes with contrasting somatic embryogenic capacity were tested using in vitro procedures. High expression of transcripts was found in embryogenic genotypes, and the results were confirmed by the RT-PCR-blotting using a non-radioactive probe. The regeneration ability was confirmed in embryogenic genotypes. We confirmed that GhSERK1 can be used as marker for estimating the somatic embryogenesis ability of cotton plants.

Risco cardiovascular: Uma visão de Indicadores de Obesidade em Pacientes Hospitalizados / Risk cardiovascular: A view of the Obesity Indicators in Hospitalized Patients

Introdução: A obesidade é um problema de saúde pública que afeta diferentes classes sociais. As pesquisas sobre obesidade vêm enfatizando, sobretudo a distribuição da gordura corporal, pois, quando a obesidade está centralizada na região abdominal as repercussões negativas são mais significativas. Objetivo: Comparar a relação cintura/estatura (RCEst) com outros indicadores de obesidade (Índice de massa corporal (IMC), Índice conicidade (IC) e Circunferência da cintura CC) a fim de identificar o indicador mais sensível para detectar risco cardiovascular em adultos e idosos internados em um Hospital Público de Sergipe. Métodos: O desenho foi de corte transversal, com amostra composta por 136 indivíduos. Foi utilizado o IC, IMC, CC e RCEst, e pontos de corte propostos pela WHO para adultos e Lipschitz para os idosos. Resultados: Os resultados encontrados mostrou que o IC possui uma área sob as curvas ROC de 0,63 e o ponto de corte foi de 1,5 (IC95% = 0,53 - 0,72) com uma sensibilidade de 97% e especificidade de 71%. A CC obteve melhor poder preditivo, com uma área total sob as curvas ROC de 0,83 para homens (IC95% = 0,76 - 0,91). O IMC mostrou-se com especificidade e sensibilidade insatisfatórios quando comparado com a RCEst. Conclusão: Os resultados demonstram que, as áreas sob a curva ROC para identificar o poder preditivo entre os indicadores antropométricos apresentam o Índice de conicidade (IC) e a circunferência da cintura (CC) como melhor discriminadora para adultos e idosos em pacientes hospitalizados considerados fatores de risco para doenças cardiometabólicos

Introduction: Obesity is a public health problem that affects different social classes. Research on obesity has been emphasizing, especially the distribution of body fat, because when obesity is centralized in the abdominal region the negative repercussions are more significant. Objective: To compare the waist/height ratio with other indicators of obesity (body mass index (BMI), conicity index (CI) and waist circumference (WC) in order to identify the most sensitive indicator to detect risk Cardiovascular disease in adults and elderly hospitalized in a Public Hospital of Sergipe. Methods: The design was cross - sectional, with a sample composed of 136 individuals. CI, BMI, WC and RCE, and cutoff points proposed by the WHO for adults and Lipschitz for the elderly were used. Results: The results showed that the CI has an area under the ROC curves of 0.63 and the cutoff point was 1.5 (95% CI = 0.53-0.72) with a sensitivity of 97% and specificity of 71 %. The WC obtained better predictive power, with a total area under the ROC curves of 0.83 for men (95% CI = 0.76-0.91). BMI showed unsatisfactory specificity and sensitivity when compared to RCE. Conclusion: The results show that the areas under the ROC curve to identify the predictive power between the anthropometric indicators present the conicity index (CI) and waist circumference (WC) as the best discriminator for adults and elderly in hospitalized patients considered as risk factors for cardiometabolic diseases

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Involvement of cytochrome P450 in cisplatin treatment: implications for toxicity.

PURPOSE: The aim of this study is to evaluate the relationship between the CYP450 enzyme family and cisplatin toxicity. METHODS: This article examined a collection of studies suggesting that CYP450 enzymes may influence cisplatin toxicity. We performed a narrative mini-review. RESULTS: The studies review showed that CYP450 enzymes have an important role in drug-induced hepatotoxicity and nephrotoxicity, mainly CYP2E1 and CYP4A11. The studies also suggested that the cisplatin and CYP2E1 interaction leads to the generation of reactive oxygen species (ROS) and other oxidants resulting in renal injury; and that ROS generated by both the use of cisplatin and by the CYP2E1 increases tissue damage, induces apoptosis, and causes liver failure. CONCLUSIONS: We observed that there is an important relationship between CYP450 and cisplatin, involving increased toxicity. However, the possible mechanisms described for the involvement of CYP450 enzymes in nephrotoxicity and hepatotoxicity induced by cisplatin need to be confirmed by further studies. Therefore, there is a need for a deeper investigation focusing on cisplatin toxicity mediated by CYP450 enzymes, which would undoubtedly contribute to a better understanding of the mechanisms that have been implicated so far.

Existe associação entre massa e força muscular esquelética em idosos hospitalizados? / Is there is an association between mass and skeletal muscle strength in hospitalized elderly persons?

Resumo Introdução: Massa e força muscular esquelética são variáveis que contribuem para o diagnóstico de sarcopenia. Objetivo: Avaliar a associação entre força e massa muscular esquelética em idosos hospitalizados. Método: Estudo transversal, realizado em hospital privado na cidade de Salvador-BA. Foram incluídos idosos ≥60 anos, entre o 1o e o 5o dia de internação hospitalar e que estivessem sem sedação e/ou drogas vasoativas. A massa muscular foi obtida por meio de equação antropométrica e a variável força por meio da força de preensão palmar. Fraqueza muscular foi identificada se <20 kgf para mulheres e <30 kgf para homens e a massa muscular reduzida quando o índice de massa muscular foi ≤8,9 kg/m2 para homens e ≤6,37 kg/m2 para mulheres. A correlação de Pearson foi utilizada para avaliar a relação entre massa e força e a acurácia para avaliar a capacidade da massa predizer força. Resultados: Entre os 110 idosos avaliados, houve moderada correlação entre massa e força (R=0,691; p=0,001). Entretanto, a acurácia foi fraca da massa para predizer força muscular (acurácia=0,30; IC 95% = 0,19-0,41; p=0,001). Os idosos com fraqueza eram mais velhos que os sem fraqueza, sem diferença nas outras variáveis. Conclusão: Existe uma relação linear entre massa e força muscular esquelética, porém a massa não prediz força, o que sugere que as duas medidas continuem sendo realizadas de forma independente.

Abstract Introduction: The variables mass and skeletal muscle strength contribute to the diagnosis of sarcopenia. Objective: To evaluate the association between strength and skeletal muscle mass in hospitalized elderly persons. Method: A cross-sectional study was carried out in a private hospital in the city of Salvador in Bahia. The study included individuals ≥60 years during their first and fifth day of hospitalization and who were neither sedated nor had taken vasoactive drugs. Muscle mass was calculated using an anthropometric equation and force was measured through handgrip strength. Muscle weakness was identified as <20 kgf for women and <30 kgf for men, and reduced muscle mass was when the muscle mass index was ≤8.9 kg/m2 for men and ≤6.37 kg/m2 for women. The Pearson correlation was used to evaluate the relationship between mass and strength and the accuracy of using mass to predict strength. Results: In 110 patients included, there was a moderate correlation between mass and strength (R=0.691; p=0.001). However, the accuracy of using mass to predict muscle strength was low (accuracy=0.30; CI 95% = 0.19-0.41; p=0.001). The elderly patients with muscle weakness were older than those without muscle weakness, with no differences between the other variables. Conclusion: There is a linear relation between skeletal muscle mass and strength, but mass did not predict strength, which suggests that the two measures continue to perform independently.

Coffee consumption, obesity and type 2 diabetes: a mini-review.

PURPOSE: The effects of regular coffee intake on weight gain and development of diabetes are reviewed. The pathophysiology of obesity and type 2 diabetes as well as the necessity of preventive options based on the increasing prevalence of these two disorders worldwide is briefly discussed. The relationship between weight gain and development of diabetes is also presented. The two major constituents in the brewed coffee, chlorogenic acids and caffeine, are responsible for many of the beneficial effects suggested by numerous epidemiological studies of coffee consumption and the development of diabetes. METHODS: A wide search of various databases, such as PubMed and Google Scholar, preceded the writing of this manuscript, focusing on key words that are part of the title. It was selected mainly review papers from in vivo, ex vivo, in vitro experimental studies in animals and human tissues as well as wide population-based epidemiological studies in the last 10 years. CONCLUSION: As of today, there are mounting evidences of the reduced risk of developing type 2 diabetes by regular coffee drinkers of 3-4 cups a day. The effects are likely due to the presence of chlorogenic acids and caffeine, the two constituents of coffee in higher concentration after the roasting process.

Stable integration and expression of a cry1Ia gene conferring resistance to fall armyworm and boll weevil in cotton plants.

BACKGROUND: Boll weevil is a serious pest of cotton crop. Effective control involves applications of chemical insecticides, increasing the cost of production and environmental pollution. The current genetically modified Bt crops have allowed great benefits to farmers but show activity limited to lepidopteran pests. This work reports on procedures adopted for integration and expression of a cry transgene conferring resistance to boll weevil and fall armyworm by using molecular tools. RESULTS: Four Brazilian cotton cultivars were microinjected with a minimal linear cassette generating 1248 putative lines. Complete gene integration was found in only one line (T0-34) containing one copy of cry1Ia detected by Southern blot. Protein was expressed in high concentration at 45 days after emergence (dae), decreasing by approximately 50% at 90 dae. Toxicity of the cry protein was demonstrated in feeding bioassays revealing 56.7% mortality to boll weevil fed buds and 88.1% mortality to fall armyworm fed leaves. A binding of cry1Ia antibody was found in the midgut of boll weevils fed on T0-34 buds in an immunodetection assay. CONCLUSION: The gene introduced into plants confers resistance to boll weevil and fall armyworm. Transmission of the transgene occurred normally to T1 progeny. All plants showed phenotypically normal growth, with fertile flowers and abundant seeds. © 2015 Society of Chemical Industry.

Adaptabilidade e estabilidade de genótipos eretos de amendoim cultivados nas regiões Nordeste e Centro-Oeste / Comparison of adaptability and stability estimates in upright peanut genotypes cultivated in Northeast and Midwest regions

O presente trabalho teve por objetivo estimar a estabilidade e a adaptabilidade de oito genótipos de amendoim ereto e comparar os modelos de análise de EBERHART & RUSSELL (1966), WRICKE (1965) e LIN & BINNS (1988), baseando-se em dados de produtividade de vagens e sementes. Os genótipos de amendoim foram avaliados em 14 ambientes situados nas Regiões Nordeste e Centro-Oeste, durante os anos de 2006 a 2011, sob o delineamento de blocos ao acaso com cinco repetições. Verificou-se que, entre os três métodos estudados, os de EBERHART & RUSSELL(1966) e LIN & BINNS (1988) geraram informações concordantes para estimativas de adaptação e estabilidade de produção de vagens e sementes, baseando-se nas condições deste estudo. Os genótipos L7 Bege e BRS 151 L7 foram os mais produtivos, com adaptabilidade específica a ambientes favoráveis, enquanto que CNPA 280 revelou adaptabilidade ampla e alta estabilidade fenotípica.

This study aimed to estimate the stability and adaptability parameters in upright-peanut genotypes, based on pod and seed yield, through EBERHART & RUSSELL (1966), WRICKE (1965) and LIN & BINNS (1988) methodologies. The genotypes were evaluated in 14 environments located in Northeast and Midwest regions, during 2006 to 2011, in a randomized blocks design, with five replications. EBERHART & RUSSELL (1966) and LIN & BINNS (1988) methodologies revealed similar results, identifying genotypes with high adaptability and stability to pod and seed yield. The BRS 151 L7 and L7 Bege were the most productive, revealing specific adaptability to favorable environment, while CNPA 280 AM revealed broad adaptability and high phenotypical stability.

Plant promoters: an approach of structure and function.

With current advances in genomics, several technological processes have been generated, resulting in improvement in different segments of molecular research involving prokaryotic and eukaryotic systems. A widely used contribution is the identification of new genes and their functions, which has led to the elucidation of several issues concerning cell regulation and interactions. For this, increase in the knowledge generated from the identification of promoters becomes considerably relevant, especially considering that to generate new technological processes, such as genetically modified organisms, the availability of promoters that regulate the expression of new genes is still limited. Considering that this issue is essential for biotechnologists, this paper presents an updated review of promoters, from their structure to expression, and focuses on the knowledge already available in eukaryotic systems. Information on current promoters and methodologies available for studying their expression are also reported.

Translocation and insecticidal activity of Bacillus thuringiensis living inside of plants.

The major biological pesticide for the control of insect infestations of crops, Bacillus thuringiensis was found to be present naturally within cotton plants from fields that had never been treated with commercial formulations of this bacterium. The ability of B. thuringiensis to colonize plants as an endophyte was further established by the introduction of a strain marked by production of green fluorescent protein (GFP). After inoculation of this preparation close to the roots of cotton and cabbage seedlings, GFP-marked bacteria could be re-isolated from all parts of the plant, having entered the roots and migrated through the xylem. Leaves taken from the treated plants were able to cause toxicity when fed to the Lepidoptera Spodoptera frugiperda (cotton) and Plutella xylostella (cabbage). These results open up new horizons for understanding the natural ecology and evolution of B. thuringiensis and use of B. thuringiensis in insect control.

Interferon-beta modulates bone-associated cytokines and osteoclast precursor activity in multiple sclerosis patients.

PURPOSE: Multiple sclerosis (MS) patients have a high risk of low bone density. The purpose of this study was to examine the molecular mechanisms potentially capable of modulating bone homeostasis in response to interferon-beta-1a (IFN-beta-1a) treatment and the focus was the bone-modulating system comprised of receptor activator of nuclear factor-kappaB (RANK), its ligand RANKL and its decoy receptor, osteoprotegerin (OPG). METHODS: In this open-label pharmacodynamic study, peripheral blood was obtained from relapsing-remitting MS patients just prior to and at multiple time points after intramuscular injection of 30 microg IFN-beta-1a. Samples were analysed for RANKL, tumour necrosis factor related apoptosis-inducing ligand (TRAIL), OPG and macrophage inflammatory protein-1 alpha/beta expression. Osteoclast precursor differentiation from peripheral blood cells of MS patients in the presence of exogenously added IFN-beta-1a was also assessed. Additionally, the changes in plasma levels of osteocalcin and the C-telopeptides after 1 year of treatment were measured as surrogate markers of bone formation and degradation, respectively. RESULTS: IFN-beta-1a treatment modulated RANKL and OPG in a selective, time-dependent manner. The levels of OPG protein decreased 25% at the 8-h time point, then increased 43% at the 24-h time point. The levels of free RANKL reached a maximum at the 8-h time point. Increases in the levels of macrophage inflammatory protein-1beta (MIP-1beta), a chemokine that increases osteolysis, were observed. The levels of the bone formation marker, osteocalcin, were lower in MS patients compared to controls and increased after one year of treatment. Ex vivo treatment of peripheral blood lymphocytes with IFN-beta resulted in a marked reduction of osteoclast-like cells in the presence of RANKL and macrophage colony stimulating factor. CONCLUSIONS: IFN-beta treatment induces complex, specific and time-dependent changes in multiple proteins and mRNAs related to bone homeostasis in MS patients.

Dynamics of interferon-beta modulated mRNA biomarkers in multiple sclerosis patients with anti-interferon-beta neutralizing antibodies.

The objective of this study was to evaluate multiple interferon (IFN) specific mRNA biomarkers in multiple sclerosis (MS) patients with anti-IFN-beta neutralizing antibodies (NAB) using a pharmacodynamic study design. Thirty patients were enrolled. Blood samples were drawn at pre-treatment, 4-, 8-h time points following the intramuscular dose of IFN-beta-1a. Total RNA was obtained from peripheral blood cells, processed to cDNA and analyzed using quantitative real-time polymerase chain reaction. Pre-treatment serum samples were analyzed for anti-IFN-beta binding and neutralizing antibodies: 22 patients were NAB negative; equal numbers of the eight remaining patients were either NAB positive or had borderline NAB status. The results showed that early assessment (at 4 h after IFN-beta injection) of mRNAs for Stat-1, MxA, MxB and TRAIL was more sensitive than the later measurements. Furthermore, the NAB positive patients had strongly attenuated gene expression responses on all the mRNAs. Patients with borderline NAB had average responses that appear to be lower than NAB negative patients on several genes, notably Stat-1, TRAIL and beta2 microglobulin.

Genomic effects of IFN-beta in multiple sclerosis patients.

The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-beta-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 micro g of IFN-beta-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-beta-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1-4 h of IFN-beta treatment. The mRNAs for several genes involved in IFN-beta signaling, such as IFN-alpha/beta receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9-27), IFN-induced transmembrane protein 2 (1-8D), beta(2)-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-beta treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.

Molecular cloning of alpha-amylases from cotton boll weevil, Anthonomus grandis and structural relations to plant inhibitors: an approach to insect resistance.

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of alpha-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two alpha-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5' and 3' RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several alpha-amylase inhibitors from plants were assayed against A. grandis alpha-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and alpha-AI1 inhibitors on A. grandis alpha-amylase activity. This work suggests that genetic engineering of cotton to express alpha-amylase inhibitors may offer a novel route to A. grandis resistance.

Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis.

The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.