Changes in landscape and climate in Mexico and Texas reveal small effects on migratory habitat of monarch butterflies (Danaus plexippus).

The decline of the iconic monarch butterfly (Danaus plexippus) in North America has motivated research on the impacts of land use and land cover (LULC) change and climate variability on monarch habitat and population dynamics. We investigated spring and fall trends in LULC, milkweed and nectar resources over a 20-year period, and ~ 30 years of climate variables in Mexico and Texas, U.S. This region supports spring breeding, and spring and fall migration during the annual life cycle of the monarch. We estimated a - 2.9% decline in milkweed in Texas, but little to no change in Mexico. Fall and spring nectar resources declined < 1% in both study extents. Vegetation greenness increased in the fall and spring in Mexico while the other climate variables did not change in both Mexico and Texas. Monarch habitat in Mexico and Texas appears relatively more intact than in the midwestern, agricultural landscapes of the U.S. Given the relatively modest observed changes in nectar and milkweed, the relatively stable climate conditions, and increased vegetation greenness in Mexico, it seems unlikely that habitat loss (quantity or quality) in Mexico and Texas has caused large declines in population size or survival during migration.

Antiproliferative activity of standardized herbal phytopreparation from Asclepias subulata.

Background: Several studies have shown that active compounds of Asclepias subulata (cardenolides) have antiproliferative effect on human cancer cells. Cardenolides isolated from A. subulata can be used as active chemical markers to elaborate phytopharmaceutical preparations. To evaluate the antiproliferative effect of a standardized extract of the aerial parts, based on Asclepias subulata cardenolides. Methods: Four standardized extracts were prepared by HPLC-DAD depending on the concentration of calotropin and the antiproliferative activity was measured for the MTT assay, on the A549, MCF-7, HeLa, PC3 and ARPE cell lines. The concentrations of calotropin used for the standardization of the extracts were 10, 7.6, 5 and 1 mg/dL. Results: Standardization of the A. subulata extract based on calotropin at 7.6 mg/g dry weight was achieved and the antiproliferative activity was evaluated over A549, HeLa and MCF-7 cell lines, obtaining proliferation percentages of 3.8 to 13.4% . Conclusions: The standardized extracts of A. subulata at different concentrations of calotropin showed antiproliferative activity against all the cell lines evaluated. The greatest effect was observed against the HeLa cell line.

Plantas tóxicas para bovinos em Minas Gerais / Toxic plants for cattle in Minas Gerais

As plantas tóxicas causam grandes prejuízos econômicos para a pecuária em diversas regiões do Brasil, provocando, até mesmo, a morte de animais. Poucas são as informações disponíveis sobre as intoxicações por plantas em bovinos, no estado de Minas Gerais. O estudo sistemático das espécies tóxicas, em regiões com poucas pesquisas, aumenta, consideravelmente, os dados sobre plantas tóxicas de interesse pecuário. Sendo assim, o objetivo deste estudo foi realizar um levantamento das plantas tóxicas para bovinos nas propriedades rurais do município de São João Evangelista, Minas Gerais. Para isso, foram visitadas, aleatoriamente, 20propriedades rurais do município e três formulários foram aplicados aos produtores rurais, sob a forma de entrevista, sendo que as informações obtidas, por meio dos formulários, foram analisadas quanti e qualitativamente. Também, foram coletadas amostras de solos para análise das características químicas do local incidente da planta tóxica. Com a análise, observou-se que a incidência de plantas tóxicas para bovinos atinge55% das propriedades rurais e em 60% das propriedades há histórico de casos de intoxicação em bovinos. Seis espécies de plantas tóxicas incidem nas pastagens das propriedades rurais, a Pteridium aquilinum, a Palicourea marcgravii, a Lantana camara, a Asclepias curassavica, a Ricinus communis e a Palicourea grandiflora. A espécie Pyrostegia venusta foi relatada como planta tóxica para bovinos e a P. marcgravii associada à maior quantidade de mortes de bovinos no município. Observou-se também, que as espécies de plantas tóxicas possuem a capacidade de desenvolvimento em condições de acidez do solo e de baixa fertilidade e em pastagens bem manejadas com boa fertilidade.(AU)

Toxic plants cause important economic damage to livestock in several regions of Brazil, even causing the death of animals. Little information is available about plant poisoning in cattle in the State of Minas Gerais. The systematic study of toxic species in regions with little research considerably increases the data on toxic plants of livestock interest. The objective of this study was to carry out a survey of toxic plants for cattle in rural properties in the municipality of São João Evangelista, Minas Gerais. Twenty rural properties in the municipality were visited at random. Three forms were applied to rural producers in the form of an interview. The information obtained through the forms were analyzed quantitatively and qualitatively. Soil samples were collected to analyze the chemical characteristics of the incident site of the toxic plant. The incidence of toxic plants for cattle affects 55% of rural properties and in 60% of properties there is a history of cases of intoxication in cattle. Six species of toxic plants affect the pastures of rural properties, being Pteridium aquilinum, Palicourea marcgravii, Lantana camara, Asclepias curassavica, Ricinus communis and Palicourea grandiflora. The species Pyrostegia venusta has been reported as a toxic plant for cattle. Palicourea marcgravii is associated with the highest number of cattle deaths in the municipality. Toxic plant species the capacity to establish themselves in conditions of soil acidity and low fertility, and in well-managed pastures with good fertility.(AU)

Plantas tóxicas para bovinos em Minas Gerais / Toxic plants for cattle in Minas Gerais

As plantas tóxicas causam grandes prejuízos econômicos para a pecuária em diversas regiões do Brasil, provocando, até mesmo, a morte de animais. Poucas são as informações disponíveis sobre as intoxicações por plantas em bovinos, no estado de Minas Gerais. O estudo sistemático das espécies tóxicas, em regiões com poucas pesquisas, aumenta, consideravelmente, os dados sobre plantas tóxicas de interesse pecuário. Sendo assim, o objetivo deste estudo foi realizar um levantamento das plantas tóxicas para bovinos nas propriedades rurais do município de São João Evangelista, Minas Gerais. Para isso, foram visitadas, aleatoriamente, 20propriedades rurais do município e três formulários foram aplicados aos produtores rurais, sob a forma de entrevista, sendo que as informações obtidas, por meio dos formulários, foram analisadas quanti e qualitativamente. Também, foram coletadas amostras de solos para análise das características químicas do local incidente da planta tóxica. Com a análise, observou-se que a incidência de plantas tóxicas para bovinos atinge55% das propriedades rurais e em 60% das propriedades há histórico de casos de intoxicação em bovinos. Seis espécies de plantas tóxicas incidem nas pastagens das propriedades rurais, a Pteridium aquilinum, a Palicourea marcgravii, a Lantana camara, a Asclepias curassavica, a Ricinus communis e a Palicourea grandiflora. A espécie Pyrostegia venusta foi relatada como planta tóxica para bovinos e a P. marcgravii associada à maior quantidade de mortes de bovinos no município. Observou-se também, que as espécies de plantas tóxicas possuem a capacidade de desenvolvimento em condições de acidez do solo e de baixa fertilidade e em pastagens bem manejadas com boa fertilidade.

Toxic plants cause important economic damage to livestock in several regions of Brazil, even causing the death of animals. Little information is available about plant poisoning in cattle in the State of Minas Gerais. The systematic study of toxic species in regions with little research considerably increases the data on toxic plants of livestock interest. The objective of this study was to carry out a survey of toxic plants for cattle in rural properties in the municipality of São João Evangelista, Minas Gerais. Twenty rural properties in the municipality were visited at random. Three forms were applied to rural producers in the form of an interview. The information obtained through the forms were analyzed quantitatively and qualitatively. Soil samples were collected to analyze the chemical characteristics of the incident site of the toxic plant. The incidence of toxic plants for cattle affects 55% of rural properties and in 60% of properties there is a history of cases of intoxication in cattle. Six species of toxic plants affect the pastures of rural properties, being Pteridium aquilinum, Palicourea marcgravii, Lantana camara, Asclepias curassavica, Ricinus communis and Palicourea grandiflora. The species Pyrostegia venusta has been reported as a toxic plant for cattle. Palicourea marcgravii is associated with the highest number of cattle deaths in the municipality. Toxic plant species the capacity to establish themselves in conditions of soil acidity and low fertility, and in well-managed pastures with good fertility.

Use of Nasoil® via intranasal to control the harmful effects of Covid-19.

In the absence of vaccines and antiviral drugs available to prevent and treat COVID-19, it becomes imperative to find or use all those products with the potential to fight this virus. This article is an attempt to propose ways to prevent, treat and control the COVID-19 virus, using a product based on plant extracts with the potential to reduce the symptoms caused by the SARS-CoV-2 virus. Nasoil® counts as one of its main components, Asclepias curassavica extracts, and in the present study it has been shown that it is an effective adjuvant in the treatment of Covid-19, increasing the respiratory capacity of the patients (SpO2> 90%) and reducing the symptoms from the first application, improving the patients around the fifth to the eighth application. At a preventive level, the individuals in this study who have applied it (400 individuals) only a 3.15% of these presented symptoms, disappearing when increasing the weekly applications.

Antimutagenic effect of an Asclepias subulata extract against heterocyclic aromatic amines commonly found in cooked meat and its heat stability.

The antimutagenicity of an extract from the medicinal plant Asclepias subulata (ASE) against heterocyclic aromatic amines (HAAs) commonly found in cooked meat, as well as its stability to heat treatment (HT), was evaluated. HT (180 °C/3 min) had no effect on the content in ASE of the bioactive compound corotoxigenin-3-O-glucopyranoside; conversely, calotropin significantly decreased by 72%. ASE exerted antimutagenicity against PhIP, MelQ, and MelQx in TA98 and TA100 Salmonella strains, and this activity was not affected by heat, with the exception of MelQ (p < 0.05). Since HAAs can induce colorectal cancer, the thermal stability of ASE's antiproliferative effect against colorectal cancer cells was also evaluated. HT decreased (p < 0.05) the antiproliferative activity of ASE; however, the remaining activity was still strong with an IC50 of 16.8 ± 2.03 µg/mL. Therefore, ASE can be used as a food ingredient to reduce the carcinogenic potential of thermally induced HAAs.

Phenolic Profile, Antioxidant and Anti-Proliferative Activities of Methanolic Extracts from Asclepias linaria Cav. Leaves.

Asclepias linaria Cav. (Apocynaceae) is a shrubby plant endemic of Mexico which has been used in traditional medicine. However, the bioactive potential of this plant remains unexplored. In this study, the phenolic composition, antioxidant, and cytotoxic activities of A. linaria leaves were determined. In order to estimate the phenolic composition of the leaves, the total phenolic, flavonoid, and condensed tannins contents were determined. Furthermore, the antioxidant activity was measured by the scavenging activity of the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS•+) radicals and the total antioxidant capacity. The phenolic compounds identified in the A. linaria leaves by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) include phenolic acids, such as p-coumaric and ferulic acid, as well as flavonoids, such as rutin and quercetin. The leaves' extracts of A. linaria showed a high scavenging activity of DPPH• and ABTS•+ radicals (IC50 0.12 ± 0.001 and 0.51 ± 0.003 µg/mL, respectively), high total antioxidant capacity values (99.77 ± 4.32 mg of ascorbic acid equivalents/g of dry tissue), and had a cytotoxic effect against K562 and HL60 hematologic neoplasia cells lines, but no toxicity towards the normal mononuclear cell line was observed. These results highlight the potential of A. linaria and could be considered as a possible alternative source of anticancer compounds.

Low level resistance of wild poinsettia biotypes to glyphosate and alternative chemical control / Resistência de nível baixo de biótipos de leiteira ao glyphosate e alternativas quimicas de controle

In the State of Rio Grande do Sul (RS), Southern Brazil, glyphosate has not been capable of controlling wild poinsettia (Euphorbia heterophylla L.) in soybean fields, thus, suggesting resistanceto this herbicide. Therefore, this study aimed at evaluating sensitivity of wild poinsettia biotypes to glyphosate, identifying the occurrence of resistance of wild poinsettia to the herbicide in RS state and determining the resistance factor of wild poinsettia biotypes under suspicion, besides assessing other herbicides as alternative controls. Two greenhouse experiments, which lasted two years, were conducted by a completely randomized design with four replications. Six biotypes (Factor A) and eight doses of glyphosate (Factor B) were used for getting the dose-response curve. Regarding the alternative control, post-emergence herbicides for soybean and corn crops were tested. Control and dry mass of the shoot were analyzed as variables. Resistance factors of resistant biotypes 20.2 and 21.1 were 4.83 and 5.29, respectively, by comparison with the susceptible biotype (11.4). In RS state, there has currently been high selection pressure due to the intensive use of glyphosate against wild poinsettia plants, as the result of the occurrence of biotypes 20.2 and 21.1 which have low levels of resistance to glyphosate and very little control by ALS-inhibiting herbicides. Therefore, an alternative to mitigate the problem is the use of herbicides with different mechanisms of action.

As falhas de controle de leiteira (Euphorbia heterophylla L.) após aplicação de glyphosate em lavouras de soja do Rio Grande do Sul (RS) são frequentes, sugerindo a resistência ao herbicida. Diante disso, os objetivos foram avaliar a sensibilidade de biótipos de leiteira ao herbicida glyphosate, identificar a ocorrência da resistência, determinar o fator de resistência de biótipos de leiteira com suspeita de resistência e avaliar herbicidas alternativos para o seu controle. Foram conduzidos dois experimentos em casa de vegetação, em delineamento inteiramente casualizado com quatro repetições ambos realizados em dois anos. No experimento de curva dose-resposta foram utilizados cinco biótipos (fator A) e oito doses do herbicidaglyphosate (fator B). Para o controle alternativo, foram testados herbicidas em pós emergência das culturas de soja e milho. As variáveis analisadas foram controle e massa seca da parte aérea. O fator de resistência dos biótipos resistentes (20.2 e 21.1) foram 4,83 e 5,29 comparativamente ao biótipo suscetível (11.4) respectivamente. Existe elevada pressão de seleção pelo glyphosate em plantas de leiteira no RS, observando-se a ocorrência de biótipos 20.2 e 21.1 com resistência de nível baixo ao herbicida e com controle reduzido pelos herbicidas inibidores de ALS. Portanto, uma alternativa para atenuar o problema é o uso de herbicidas com diferentes mecanismos de ação

Interpreting surveys to estimate the size of the monarch butterfly population: Pitfalls and prospects.

To assess the change in the size of the eastern North American monarch butterfly summer population, studies have used long-term data sets of counts of adult butterflies or eggs per milkweed stem. Despite the observed decline in the monarch population as measured at overwintering sites in Mexico, these studies found no decline in summer counts in the Midwest, the core of the summer breeding range, leading to a suggestion that the cause of the monarch population decline is not the loss of Midwest agricultural milkweeds but increased mortality during the fall migration. Using these counts to estimate population size, however, does not account for the shift of monarch activity from agricultural fields to non-agricultural sites over the past 20 years, as a result of the loss of agricultural milkweeds due to the near-ubiquitous use of glyphosate herbicides. We present the counter-hypotheses that the proportion of the monarch population present in non-agricultural habitats, where counts are made, has increased and that counts reflect both population size and the proportion of the population observed. We use data on the historical change in the proportion of milkweeds, and thus monarch activity, in agricultural fields and non-agricultural habitats to show why using counts can produce misleading conclusions about population size. We then separate out the shifting proportion effect from the counts to estimate the population size and show that these corrected summer monarch counts show a decline over time and are correlated with the size of the overwintering population. In addition, we present evidence against the hypothesis of increased mortality during migration. The milkweed limitation hypothesis for monarch decline remains supported and conservation efforts focusing on adding milkweeds to the landscape in the summer breeding region have a sound scientific basis.

Validación del uso tradicional de especies de asclepias contra el veneno de bothrops diporus (yarará chica) en el nordeste de Argentina / Validation of the traditional use of asclepias species against bothrops diporus (yarará chica) venom in northeast of Argentina

La familia Asclepiadaceae posee tradición en etnomedicina. En el nordeste argentino, A.mellodora y A. curassavica se utilizan como cataplasmas en accidentes de ofidios. En este trabajo, los extractos acuosos, etanólicos y hexánicos de A. mellodora y A. curassavica se evaluaron por SDS-PAGE para determinar su actividad alexitérica. El estudio in vitro de la capacidad inhibitoria de las actividades proteolítica, hemolítica indirecta y coagulante permitieron determinar que ambas especies manifiestan actividad, siendo A. mellodora más activa. Estadísticamente los extractos de A. mellodora fueron igualmente activos contra el veneno de Bothrops diporus y no mostraron diferencias significativas respecto del órgano utilizado en la inhibición de la actividad coagulante. Este resultado está en consonancia con la forma tradicional de su uso como cataplasma. Sobre el extracto etanólico de las raíces de A. mellodora se realizó un fraccionamiento bioguiado que permitió identificar fracciones de compuestos responsables de la actividad.

The Aclepiadaceae family has been reported by its use in ethnomedicine. In the northeast of Argentina, A. mellodora and A. curassavica are traditionally used in ofidic accidents as poultices. In this work, aqueous, alcoholic and hexanoic extracts were analyzed by SDS-PAGE to determine their anti-snake activity. The in vitro study of the inhibitory ability of the following activities: proteolytic, indirect hemolytic activity and inhibition of the coagulant activity, allowed demonstrating that both species were active against venom, being A. mellodora the most active. Statistically, all extracts of A. mellodora were active against venom in the inhibition of the coagulant activity, without significant differences with respect to the organ used; which is consistent with the traditional use as external poultice. The alcoholic extract of A. mellodora roots was subjected to a bio-guided separation. The fractions obtained were enriched in compounds which could probably be responsible for the activity against venom.

Climate change may alter breeding ground distributions of eastern migratory monarchs (Danaus plexippus) via range expansion of Asclepias host plants.

Climate change can profoundly alter species' distributions due to changes in temperature, precipitation, or seasonality. Migratory monarch butterflies (Danaus plexippus) may be particularly susceptible to climate-driven changes in host plant abundance or reduced overwintering habitat. For example, climate change may significantly reduce the availability of overwintering habitat by restricting the amount of area with suitable microclimate conditions. However, potential effects of climate change on monarch northward migrations remain largely unknown, particularly with respect to their milkweed (Asclepias spp.) host plants. Given that monarchs largely depend on the genus Asclepias as larval host plants, the effects of climate change on monarch northward migrations will most likely be mediated by climate change effects on Asclepias. Here, I used MaxEnt species distribution modeling to assess potential changes in Asclepias and monarch distributions under moderate and severe climate change scenarios. First, Asclepias distributions were projected to extend northward throughout much of Canada despite considerable variability in the environmental drivers of each individual species. Second, Asclepias distributions were an important predictor of current monarch distributions, indicating that monarchs may be constrained as much by the availability of Asclepias host plants as environmental variables per se. Accordingly, modeling future distributions of monarchs, and indeed any tightly coupled plant-insect system, should incorporate the effects of climate change on host plant distributions. Finally, MaxEnt predictions of Asclepias and monarch distributions were remarkably consistent among general circulation models. Nearly all models predicted that the current monarch summer breeding range will become slightly less suitable for Asclepias and monarchs in the future. Asclepias, and consequently monarchs, should therefore undergo expanded northern range limits in summer months while encountering reduced habitat suitability throughout the northern migration.

Unravelling the annual cycle in a migratory animal: breeding-season habitat loss drives population declines of monarch butterflies.

Threats to migratory animals can occur at multiple periods of the annual cycle that are separated by thousands of kilometres and span international borders. Populations of the iconic monarch butterfly (Danaus plexippus) of eastern North America have declined over the last 21 years. Three hypotheses have been posed to explain the decline: habitat loss on the overwintering grounds in Mexico, habitat loss on the breeding grounds in the United States and Canada, and extreme weather events. Our objectives were to assess population viability, determine which life stage, season and geographical region are contributing the most to population dynamics and test the three hypotheses that explain the observed population decline. We developed a spatially structured, stochastic and density-dependent periodic projection matrix model that integrates patterns of migratory connectivity and demographic vital rates across the annual cycle. We used perturbation analysis to determine the sensitivity of population abundance to changes in vital rate among life stages, seasons and geographical regions. Next, we compared the singular effects of each threat to the full model where all factors operate concurrently. Finally, we generated predictions to assess the risk of host plant loss as a result of genetically modified crops on current and future monarch butterfly population size and extinction probability. Our year-round population model predicted population declines of 14% and a quasi-extinction probability (<1000 individuals) >5% within a century. Monarch abundance was more than four times more sensitive to perturbations of vital rates on the breeding grounds than on the wintering grounds. Simulations that considered only forest loss or climate change in Mexico predicted higher population sizes compared to milkweed declines on the breeding grounds. Our model predictions also suggest that mitigating the negative effects of genetically modified crops results in higher population size and lower extinction risk. Recent population declines stem from reduction in milkweed host plants in the United States that arise from increasing adoption of genetically modified crops and land-use change, not from climate change or degradation of forest habitats in Mexico. Therefore, reducing the negative effects of host plant loss on the breeding grounds is the top conservation priority to slow or halt future population declines of monarch butterflies in North America.

The buck in the milkweed: evidence of male-male interference among pollinaria on pollinators.

Direct physical confrontation among conspecifics for access to mates is a form of sexual selection well known among animals, but not thought to take place in plants. Consequently, no structures are known that can be considered as weapons that evolved under such confrontation. Pollinaria of milkweeds may physically compete for access to attachment points on the pollinators' body, and occasionally pollinaria may link onto pre-existing pollinaria on a pollinator resulting in concatenation. We hypothesized that concatenation may result in interference between proximal and distal pollinaria, and that features of nonconcatenating pollinaria might be attributed to prevention of concatenation. We tested this by analyzing pollen donation efficiency, experimental manipulation of the phenotype and the phylogenetic patterns of co-occurrence of traits. It is shown that concatenation was able to diminish the reproductive performance of proximal pollinaria, that horns on pollinaria prevented concatenation, and that horn acquisition was correlated with a loss of concatenation. The experimental removal of horns in species that did not concatenate caused reversion to concatenation. The present work could be the first evidence of male physical struggles and of the acquisition of weapons related to these struggles, that are analogous to those known in animals.

Diagnose of the ingestion of Asclepias mellodora St. Hil. by sheep through microhistological analysis of their digestive contents / Diagnose da ingestão de Asclepias mellodora St. Hil. por ovinos através da análise micro-histológica de seu conteúdo digestivo

Asclepias mellodora St. Hil. is a native acute toxic species frequent in the grasslands of the Buenos Aires province, Argentina, whose toxicity had not been assessed until now. This study evaluates the minimal lethal dose of this species for sheep, and the possibility of microscopically recognizing its fragments in gastrointestinal contents as a complementary diagnostic tool in necropsies. Three Frisona sheep (average LW=55±4.5 kg) were dosed via an esophageal tube with each one of the following doses of asclepias: 8.0, 5.0, 2.0 and 0.8 g DM.kg LW-1. Sheep poisoned with the three higher doses died between 10 and 85 h after intoxication, but those receiving the lower dose did not. During necropsies we: 1) determined the dry weight of the contents of rumen+reticulum, omasum+abomasum, and large intestine, 2) estimated the percentages of asclepias fragments by microanalysis correcting for digestion effects on fragment recognition, and 3) calculated the total mass of asclepias in the digestive tract of each animal. For the three higher doses, the mass of asclepias identified in the total ingesta was 12.3±3.4 percent of the amount supplied, possibly because of the strong diarrhea its ingestion produced. The percentages of asclepias in rumen+reticulum did not differ from the average quantified for the entire tract. The results of this study indicate that the minimal lethal doses of asclepias for sheep is between 2.0 and 0.8g DMÀkg LW-1, and that the microhistological analysis of the rumen+reticulum, the easiest region to sample, can be used to confirm the ingestion of this toxic species, although the estimated percentage will be not a good estimator of the ingested percentage.(AU)

Asclepias mellodora St. Hil. é uma espécie nativa de aguda toxicidade, frequente nos campos da província de Buenos Aires, Argentina. A sua toxicidade não foi avaliada até agora. Este estudo avalia a dose mínima letal desta espécie, para os ovinos, bem como a possibilidade de reconhecer microscopicamente seus fragmentos no conteúdo gastrointestinal como uma ferramenta complementar de diagnóstico em necropsias. Três ovinos Frisona (PV média = 55±4,5 kg) foram dosados através de uma sonda esofágica em cada uma das seguintes doses de Asclepias: 8,0, 5,0, 2,0 e 0,8 g DM.kg PV-1. Ovinos intoxicados com as três maiores doses morreram entre 10-85 h após a intoxicação, mas não aqueles que receberam a dose menor. Durante as necropsias se: 1) determinou o peso seco do conteúdo do rúmen + retículo, omaso + abomaso e intestino grosso, 2) estimou as porcentagens de fragmentos de Asclepias por microanálise, fazendo a correção para efeitos de digestão no reconhecimento dos fragmentos, e 3) calculou a massa total de Asclepias no trato digestivo de cada animal. Para as três doses maiores, a massa de Asclepias identificada na ingesta total foi de 12,3±3,4 por cento da quantidade fornecida, possivelmente por causa da forte diarréia produzida pela sua ingestão. As porcentagens de Asclepias no rúmen + retículo não diferiram da média quantificada para o trato completo. Os resultados deste estudo indicam que a dose letal mínima de Asclepias em ovinos é de entre 2,0 e 0,8 g kg PV ò DM-1, e que a análise micro-histológica do rúmen + retículo, a região mais fácil de amostrar, pode ser usada para confirmar a ingestão desta espécie tóxica, embora a percentagem estimada não será um bom estimador da porcentagem ingerida.(AU)

Diagnose of the ingestion of Asclepias mellodora St. Hil. by sheep through microhistological analysis of their digestive contents / Diagnose da ingestão de Asclepias mellodora St. Hil. por ovinos através da análise micro-histológica de seu conteúdo digestivo

Asclepias mellodora St. Hil. is a native acute toxic species frequent in the grasslands of the Buenos Aires province, Argentina, whose toxicity had not been assessed until now. This study evaluates the minimal lethal dose of this species for sheep, and the possibility of microscopically recognizing its fragments in gastrointestinal contents as a complementary diagnostic tool in necropsies. Three Frisona sheep (average LW=55±4.5 kg) were dosed via an esophageal tube with each one of the following doses of asclepias: 8.0, 5.0, 2.0 and 0.8 g DM.kg LW-1. Sheep poisoned with the three higher doses died between 10 and 85 h after intoxication, but those receiving the lower dose did not. During necropsies we: 1) determined the dry weight of the contents of rumen+reticulum, omasum+abomasum, and large intestine, 2) estimated the percentages of asclepias fragments by microanalysis correcting for digestion effects on fragment recognition, and 3) calculated the total mass of asclepias in the digestive tract of each animal. For the three higher doses, the mass of asclepias identified in the total ingesta was 12.3±3.4 percent of the amount supplied, possibly because of the strong diarrhea its ingestion produced. The percentages of asclepias in rumen+reticulum did not differ from the average quantified for the entire tract. The results of this study indicate that the minimal lethal doses of asclepias for sheep is between 2.0 and 0.8g DMÀkg LW-1, and that the microhistological analysis of the rumen+reticulum, the easiest region to sample, can be used to confirm the ingestion of this toxic species, although the estimated percentage will be not a good estimator of the ingested percentage.

Asclepias mellodora St. Hil. é uma espécie nativa de aguda toxicidade, frequente nos campos da província de Buenos Aires, Argentina. A sua toxicidade não foi avaliada até agora. Este estudo avalia a dose mínima letal desta espécie, para os ovinos, bem como a possibilidade de reconhecer microscopicamente seus fragmentos no conteúdo gastrointestinal como uma ferramenta complementar de diagnóstico em necropsias. Três ovinos Frisona (PV média = 55±4,5 kg) foram dosados através de uma sonda esofágica em cada uma das seguintes doses de Asclepias: 8,0, 5,0, 2,0 e 0,8 g DM.kg PV-1. Ovinos intoxicados com as três maiores doses morreram entre 10-85 h após a intoxicação, mas não aqueles que receberam a dose menor. Durante as necropsias se: 1) determinou o peso seco do conteúdo do rúmen + retículo, omaso + abomaso e intestino grosso, 2) estimou as porcentagens de fragmentos de Asclepias por microanálise, fazendo a correção para efeitos de digestão no reconhecimento dos fragmentos, e 3) calculou a massa total de Asclepias no trato digestivo de cada animal. Para as três doses maiores, a massa de Asclepias identificada na ingesta total foi de 12,3±3,4 por cento da quantidade fornecida, possivelmente por causa da forte diarréia produzida pela sua ingestão. As porcentagens de Asclepias no rúmen + retículo não diferiram da média quantificada para o trato completo. Os resultados deste estudo indicam que a dose letal mínima de Asclepias em ovinos é de entre 2,0 e 0,8 g kg PV ò DM-1, e que a análise micro-histológica do rúmen + retículo, a região mais fácil de amostrar, pode ser usada para confirmar a ingestão desta espécie tóxica, embora a percentagem estimada não será um bom estimador da porcentagem ingerida.

Characterization of papain-like isoenzymes from latex of Asclepias curassavica by molecular biology validated by proteomic approach.

Latices from Asclepias spp are used in wound healing and the treatment of some digestive disorders. These pharmacological actions have been attributed to the presence of cysteine proteases in these milky latices. Asclepias curassavica (Asclepiadaceae), "scarlet milkweed" is a perennial subshrub native to South America. In the current paper we report a new approach directed at the selective biochemical and molecular characterization of asclepain cI (acI) and asclepain cII (acII), the enzymes responsible for the proteolytic activity of the scarlet milkweed latex. SDS-PAGE spots of both purified peptidases were digested with trypsin and Peptide Mass Fingerprints (PMFs) obtained showed no equivalent peptides. No identification was possible by MASCOT search due to the paucity of information concerning Asclepiadaceae latex cysteine proteinases available in databases. From total RNA extracted from latex samples, cDNA of both peptidases was obtained by RT-PCR using degenerate primers encoding Asclepiadaceae cysteine peptidase conserved domains. Theoretical PMFs of partial polypeptide sequences obtained by cloning (186 and 185 amino acids) were compared with empirical PMFs, confirming that the sequences of 186 and 185 amino acids correspond to acI and acII, respectively. N-terminal sequences of acI and acII, characterized by Edman sequencing, were overlapped with those coming from the cDNA to obtain the full-length sequence of both mature peptidases (212 and 211 residues respectively). Alignment and phylogenetic analysis confirmed that acI and acII belong to the subfamily C1A forming a new group of papain-like cysteine peptidases together with asclepain f from Asclepias fruticosa. We conclude that PMF could be adopted as an excellent tool to differentiate, in a fast and unequivocal way, peptidases with very similar physicochemical and functional properties, with advantages over other conventional methods (for instance enzyme kinetics) that are time consuming and afford less reliable results.

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex.

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.

Biochemical analysis of a papain-like protease isolated from the latex of Asclepias curassavica L.

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQCGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-alpha-CBZ-L-Gln-p-nitrophenyl ester as substrate: K(m)=0.1634 mM, k(cat)=121.48 s(-1), and k(cat)/K(m)=7.4 x 10(5) s(-1)/mM.

Purification and biochemical characterization of asclepain c I from the latex of Asclepias curassavica L.

In this work we report the isolation, purification and characterization of a new protease from latex of Asclepias curassavica L. Crude extract (CE) was obtained by gathering latex on 0.1 M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (km and Kcat) were performed with PFLNA.