Phytotoxic Activity of Parthenium Against Wheat and Canola Differ With Plant Parts and Bioassays Techniques

Phytotoxic effects of invasive weed Parthenium hysterophorus were studied by using whole plant, leaf and root aqueous extracts at 0, 2.5, 5.0, 7.5 and 10% (w/v) concentrations against germination and early seedling growth of wheat and canola. Studies were carried out both in Petri plates with filter paper as substratum placed in controlled conditions and soil-filled plastic pots placed in open environments. Pronounced variation was noted for phytotoxic activity of different plant parts of parthenium, aqueous extract concentrations, test species, and bioassay techniques. Aqueous parthenium extracts either inhibited or delayed the germination and suppressed seedling growth of test species over control. For both test species, all the germination attributes were suppressed to a greater extent in Petri plates than in plastic pots. Leaf extracts were more suppressive to germination of test species than whole plant and root extracts. Increasing extract concentration beyond 2.5% caused significant reduction in seedling dry biomass of both test species. Aqueous parthenium extract diminished chlorophyll contents of wheat and canola by 32-63% and 29 69%, respectively. Nevertheless, an increase of 9-172% and 22-60% in phenolic contents of wheat and canola was recorded. Canola appeared to be more susceptible than wheat at all extract concentrations. Present study concluded that bioassays conducted under controlled condition using filter paper as substratum may be misleading due to over estimation of allelopathic response and variation in potential of receiver and donor species. Furthermore, it implies that threshold concentrations of allelochemicals for test species in Petri plates are rarely reached under field conditions.

Os produtos naturais e a química medicinal moderna

Natural products have been utilized by humans since ancient times and the relief and cure of their diseases was the first purpose for using natural products in medicine. The history of the oriental and occidental civilizations is very rich in examples of the utilization of natural products in medicine and health care. Chinese traditional medicine is one of the most important examples of how natural products can be efficient in the treatment of diseases, and it points to the importance of scientific research on natural products, concerning the discovery of new active chemical entities. The complexity, chemical diversity and biological properties of natural products always fascinated people, and during the last 200 years, this led to the discovery of important new drugs. In the last 30 years, the development of new bioassay techniques, biotechnology methods, bio-guided phytochemical studies, automated high throughput screening and high performance analytical methods, have introduced new concepts and possibilities of rational drug design and drug discovery. In this context, natural products have played an important and decisive role in the development of modern medicinal chemistry.

The effects of static and diurnally-cycling temperature acclimations on thermal tolerances of rainbow trout, Salmo gairdneri

:ofiedian lethal temperatures ( LT50' s ) were determined for rainbow trout, Salmo gairdnerii, acclimated for a minimum of 21 days at 5 c onstant temperatures between 4 and 20 0 C. and 2 diel temperature fluctuations ( sinewave curves of amplitudes ± 4 and ± 7 0 C. about a mean temperature of 12 0 C. ) . Twenty-four-, 48-, and 96-hour LT50 estimates were c alculated f ollowing standard flow-through aquatic bioassay techniques and probi t transformation of mortality data. The phenomenon of delayed thermal mortality was also investigated. Shifts in upper incipient lethal temperature occurred as a result of previous thermal conditioning. It was shown that increases in constant acclimation temperature result in proportional l inear increases in thermal tolerances. The increase i n estimated 96-hour LT50's was approximately 0.13 0 c. X 1 0 C:1 between 8 and 20 0 C. The effect of acclimation to both cyclic temperature regimes was an increase in LT50 to values between the mean and maximum constant equivalent daily temperatures of the cycles. Twenty-four-, 48-, and 96-hour LT50 estimates of both cycles corresponded approximately to the LT50 values of the 16 0 C. c onstant temperature equivalent . This increase i n thermal tolerance was further demonstrated by the delayed thermal mortality experiments . Cycle amplitudes appeared to i nfluence thermal resistance through alterations in initi al mortality since mortality patterns characteristic of base temperature acclimations re-appeared after approximately 68 hours exposure to test temperatures for the 12 + 4 0 C. group, whereas mortality patterns stabilized and remained constant for a period greater than 192 hours with the larger therma l cycle ( 12 + 7 0 C. ). NO s ignificant corre lations between s pecimen weight and time-to-death was apparent. Data are discussed in relation to the establishment of thermal criteria for important commercial and sport fishes , such as the salmonids , as is the question whether previously reported values on lethal temperature s may have been under estimated.

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) resistance to fipronil in Uruguay evaluated by in vitro bioassays

Rhipicephalus (Boophilus) microplus obtained from four local populations in Uruguay (2007-2008) were subjected to various bioassay techniques to determine the presence of fipronil resistance within the country. Resistance ratios (RRs) obtained by larval immersion test varied between 3.3 and 3635 for tick populations subjected to treatment with fipronil for the last 3-7 years. The highest RR was observed in the population which received fewer treatments. Using discriminating concentration (8 ppm) for larval immersion test, all field strains were correctly diagnosed as fipronil-resistant. This study presents the first diagnoses of cattle tick resistance to fipronil in Uruguay`s field populations. It also highlights the importance of the possible conflict between programs to control agricultural pests and cattle ticks. The findings provide valuable information for selection and adoption of new control alternatives to manage drug resistance exhibited by cattle ticks. (C) 2009 Elsevier BM. All rights reserved.

Detection of Toxoplasma gondii oocysts in environmental samples from public schools

The number of Toxoplasma gondii oocysts that can be found in random environmental samples is probably low; in addition, these cysts may be confused with Hammondia spp. and Neospora spp. oocysts. The aim of the present work was to evaluate the presence of T. gondii oocysts in the soil of public elementary schools in the northwest area of the state of São Paulo, Brazil using mouse bioassays. A comparison was made between the different available bioassay techniques, such as squash, histopathology, immunohistochemistry and indirect fluorescent antibody test (IFAT). T. gondii was isolated by bioassay in mice (squash brain samples) from 22.58%(7/31) of the school playgrounds. Immunohistochemistry and IFAT showed positive results in 32.26% (10/31) and 25.80% (8/31) of samples, respectively. The sensitivity and specificity of the immunohistochemistry method were 85.71% and 83.33%, respectively. The IFAT results showed 100% sensitivity and 95.83% specificity. The presence of T. gondii was not detected in histopathological examinations. The results of the present study strongly suggest that T. gondii oocysts are widely distributed in elementary public schools in the region that was evaluated, likely constituting the main contamination source for these children. Educational programs directed at reducing environmental contamination with T. gondii would eventually lower the cost of treating humans for clinical toxoplasmosis. It is also possible to conclude that the use of IFAT in mouse bioassays can be recommended without the need for brain cysts research, which is extremely difficult and laborious. (C) 2010 Elsevier B.V. All rights reserved.

Toxoplasma gondii: Evidence for the transmission by semen in dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

OS produtos naturais e a química medicinal moderna

Natural products have been utilized by humans since ancient times and the relief and cure of their diseases was the first purpose for using natural products in medicine. The history of the oriental and occidental civilizations is very rich in examples of the utilization of natural products in medicine and health care. Chinese traditional medicine is one of the most important examples of how natural products can be efficient in the treatment of diseases, and it points to the importance of scientific research on natural products, concerning the discovery of new active chemical entities. The complexity, chemical diversity and biological properties of natural products always fascinated people, and during the last 200 years, this led to the discovery of important new drugs. In the last 30 years, the development of new bioassay techniques, biotechnology methods, bio-guided phytochemical studies, automated high throughput screening and high performance analytical methods, have introduced new concepts and possibilities of rational drug design and drug discovery. In this context, natural products have played an important and decisive role in the development of modern medicinal chemistry.

Transmissão sexual do Toxoplasma gondii (Nicolle & Manceaux, 1909) em caprinos (Capra hircus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aspects of toxoplasma infection on the reproductive system of experimentally infected rams (ovis aries)

Eight reproductive rams with no prior reproductive disease were distributed into three groups of infection with T. gondii: GI, 3 rams, 2.0 x 10(5) P strain oocysts; GII, 3 rams, 1.0 x 10(6) RH strain tachyzoites; GIII, 2 control rams. Clinical parameters were measured and serological evaluations (IIF) were performed. Presence of the parasite in the semen was investigated by PCR and bioassay techniques. The rams presented clinical alterations (hyperthermia and apathy) related to toxoplasmosis in both groups infected with Toxoplasma gondii. All the inoculated rams responded to antigenic stimulus, producing antibodies against T. gondii from postinoculation day 5 onwards. In ovine groups I and II, the greatest titers observed were 1 : 4096 and 1 : 8192, respectively. In semen samples collected from these two groups, the presence of T. gondii was detected by bioassay and PCR. This coccidian was isolated (bioassay and PCR) in tissue pools (testicles, epididymis, seminal vesicle, and prostrate) from two rams infected presenting oocysts and in one presenting tachyzoites.

Limitations on bioassays in macronutrient deficiency determination

Short-term nutrient bioassays can be used to assess labile nutrient availability in soils. These bioassays rely on a high number of plants and small soil volumes to exploit labile soil resources rapidly and assess potential nutrient deficiency. A comparison of the Neubauer bioassay with conventional pot trial assessment of P and S availability in a Yellow Kurosol was undertaken. Changes in labile soil nutrients and enzyme activity after bioassay assessment were also measured. The Neubauer bioassay was able to detect increased labile P availability following P fertiliser application to the soil. This corresponded with response to added P in a longer-term pot trial using maize. As expected, phosphatase activity increased following the bioassay and labile P was depleted by the plants. However, although a longer-term pot trial demonstrated the Yellow Kurosol was responsive to S fertilisation, labile S pools were sufficiently large that the short-term Neubauer bioassay detected no difference in S availability to plants. Both soil sulphatase activity and labile soil S were elevated following the bioassay. The short period of contact between the roots of the bioassay and the soil may have limited S uptake and therefore the ability of the bioassay to identify a S responsive soil. When using bioassay techniques to assess labile nutrient availability, it is critical that the size of the labile nutrient pool present be considered for each element, and that the period of contact between the bioassay and soil being tested is long enough for plant uptake to lower the nutrient supply to a level that limits further uptake.

Variability of voriconazole plasma levels measured by new high-performance liquid chromatography and bioassay methods, [and] Voriconazole therapeutic drug monitoring in patients with invasive mycoses improves efficacy and safety outcomes

Rapport de synthèse1. Partie de laboratoireCette première étude décrit le développement et la validation, selon les standards internationaux, de deux techniques de mesure des concentrations sanguines de voriconazole, un nouvel agent antifongique à large spectre: 1) la chromatographic en phase liquide à haute pression et 2) le bio-essai utilisant une souche mutante de Candida hypersensible au voriconazole. Ce travail a aussi permis de mettre en évidence une importante et imprévisible variabilité inter- et intra-individuelle des concentrations sanguines de voriconazole malgré l'utilisation des doses recommandées par le fabriquant. Ce travail a été publié dans un journal avec "peer-review": "Variability of voriconazole plasma levels measured by new high- performance liquid chromatography and bioassay methods" by A. Pascual, V. Nieth, T. Calandra, J. Bille, S. Bolay, L.A. Decosterd, T. Buclin, P.A. Majcherczyk, D. Sanglard, 0. Marchetti. Antimicrobial Agents Chemotherapy, 2007; 51:137-432. Partie CliniqueCette deuxième étude a évalué de façon prospective l'impact clinique des concentrations sanguines de voriconazole sur l'efficacité et sécurité thérapeutique chez des patients atteints d'infections fongiques. Des concentrations sanguines élevées étaient significativement associés à la survenue d'une toxicité neurologique (encéphalopathie avec confusion, hallucinations et myoclonies) et des concentrations sanguines basses à une réponse insuffisante au traitement antifongique (persistance ou progression des signes cliniques et radiologiques de l'infection). Dans la majorité des cas, un ajustement de la dose de voriconazole, sur la base des concentrations mesurées, a abouti à une récupération neurologique complète ou à une résolution de l'infection, respectivement. Ce travail a été publié dans un journal avec "peer-review": " Voriconazole Therapeutic Drug Monitoring in Patients with Invasive Mycoses Improves Efficacy and Safety Outcomes" by A. Pascual, T. Calandra, S. Bolay, T. Buclin, J. Bille, and O. Marchetti. Clinical Infectious Diseases, 2008 January 15; 46(2): 201-11.Ces deux études, financées de façon conjointe par un "grant" international de la Société suisse d'infectiologie et la Société internationale de maladies infectieuses et par la Fondation pour le progrès en microbiologie médicale et maladies infectieuses (FAMMID, Lausanne), ont été réalisées au sein du Service des Maladies Infectieuses, Département de Médecine, au CHUV, en étroite collaboration avec la Division de Pharmacologie Clinique, Département de Médecine, au CHUV et l'Institut de Microbiologie du CHUV et de l'Université de Lausanne.

Ultra-performance liquid chromatography mass spectrometry and sensitive bioassay methods for quantification of posaconazole plasma concentrations after oral dosing.

Posaconazole (POS) is a new antifungal agent for prevention and therapy of mycoses in immunocompromised patients. Variable POS pharmacokinetics after oral dosing may influence efficacy: a trough threshold of 0.5 ?g/ml has been recently proposed. Measurement of POS plasma concentrations by complex chromatographic techniques may thus contribute to optimize prevention and management of life-threatening infections. No microbiological analytical method is available. The objective of this study was to develop and validate a new simplified ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method and a sensitive bioassay for quantification of POS over the clinical plasma concentration range. The UPLC-MS/MS equipment consisted of a triple quadrupole mass spectrometer, an electrospray ionization (ESI) source, and a C(18) analytical column. The Candida albicans POS-hypersusceptible mutant (MIC of 0.002 ?g/ml) ?cdr1 ?cdr2 ?flu ?mdr1 ?can constructed by targeted deletion of multidrug efflux transporters and calcineurin genes was used for the bioassay. POS was extracted from plasma by protein precipitation with acetonitrile-methanol (75%/25%, vol/vol). Reproducible standard curves were obtained over the range 0.014 to 12 (UPLC-MS/MS) and 0.028 to 12 ?g/ml (bioassay). Intra- and interrun accuracy levels were 106% ± 2% and 103% ± 4% for UPLC-MS/MS and 102% ± 8% and 104% ± 1% for bioassay, respectively. The intra- and interrun coefficients of variation were 7% ± 4% and 7% ± 3% for UPLC-MS/MS and 5% ± 3% and 4% ± 2% for bioassay, respectively. An excellent correlation between POS plasma concentrations measured by UPLC-MS/MS and bioassay was found (concordance, 0.96). In 26 hemato-oncological patients receiving oral POS, 27/69 (39%) trough plasma concentrations were lower than 0.5 ?g/ml. The UPLC-MS/MS method and sensitive bioassay offer alternative tools for accurate and precise quantification of the plasma concentrations in patients receiving oral posaconazole.

Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free ranges chickens from Pantanal of Mato Grosso do Sul

The aim of this study was to investigate the occurrence of Toxoplasma gondii and compare the results obtained in the Modified Agglutination Test (MAT), Polimerase Chain Reaction (PCR) and bioassay in mice. In order to accomplish this, 40 free-range chickens from eight farms in neighboring areas to the Pantanal in Nhecolândia, Mato Grosso do Sul, were euthanized and blood samples, brain and heart were collected. The occurrence of anti-T. gondii antibodies found in chickens was 67.5% (27 samples), considering as a cutoff point the dilution 1:5. Among the samples analyzed, 7 (25.9%) were positive in the dilution 1:5, 3 (11.1%) in 1:10, 2 (7.4%) in 1:20, 3 (11.1%) in 1:320, 1 ( 3.7%) in 1:640, 3 (11.1%) in 1:1280, 2 (7.4%) in 1:2560, 4 (14.8%) in 1:5120 and 2 (7.4%) in 1:10.240. From the mixture of tissue samples (brain and heart) from the chickens analyzed, 16 (40%) presented electrophoretic bands compatible with T. gondii by PCR (gene B1). In the comparison of techniques, 59.26% positivity in PCR was revealed among animals that were seropositive in MAT (cutoff 1:5). From 141 inoculated mice, six (4.44%) died of acute toxoplasmosis between 15 and 23 days after inoculation. Surviving mice were sacrificed at 74 days after inoculation, and a total of 28 cysts were found in the brains of 10 distinct groups. From the seropositive hens, 27 bioassays were performed and 11 (40.7%) isolates were obtained. A greater number of isolations happened in mice that were inoculated with tissues from chickens that had high titers for anti-T. gondii antibodies. Chronic infection in mice was observed in nine groups (33.3%) from five different properties. Among the surviving mice, 25.6% were positive for T. gondii in MAT (1:25). From mice positive in PCR, 87.5% were also positive in MAT. Among the PCR-negative mice, 5.2% were positive for T. gondii in MAT. It can be concluded through this study that the occurrence of infecton by T. gondii in the rural properties studied was high, that PCR directed to gene B1 does not confirm the viability of the parasite, but it can be used as a screening method for the selection of chickens infected by T. gondii, that the animals with titer greater than 10 must be prioritized for the selection of animals for bioassay, since for them, the chances of isolating the parasite are greater and that seroconversion in experimentally infected mice is not a good indicator for isolating the agent.

Toxoplasma gondii: Detection by mouse bioassay, histopathology, and polymerase chain reaction in tissues from experimentally infected pigs

In the present study, we evaluated three techniques, mouse bioassay, histopathology, and polymerase chain reaction (PCR) to detect Toxoplasma gondii infection in tissues from experimentally infected pigs. Twelve mixed breed pigs, seronegative for T. gondii using an indirect immunofluorescent antibody test (IFAT), were used. Ten pigs were infected with 4 × 104 VEG strain oocysts, and two were maintained as uninfected controls. Animals were killed 60 days pos infection. Muscle (heart, tongue, diaphragm, and masseter) and brain samples were collected to investigate the presence of T. gondii tissue cysts by the different assay methods. For the bioassay, samples of brain (50 g) and pool of muscle samples (12.5 g of tongue, masseter, diaphragm, and heart) were used. PCR was performed using Tox4 and Tox5 primers which amplified a 529 bp fragment. The DNA extraction and PCR were performed three times, and all tissue samples were tested individually (brain, tongue, masseter, diaphragm, and heart). For histopathology, fragments of tissues were fixed in 10% of buffered formal saline and stained with HE. Histopathological results were all negative. PCR showed 25/150 (16.6%) positive samples, being 17/120 (14.1%) and 8/30 (26.6%) from muscle, and brain tissues, respectively. Tissue cysts of T. gondii were identified by mouse bioassay in 54/98 (55.1%) samples, being 31/48 (64.6%) from muscle samples, and 23/50 (46.0%) from brain samples. Toxoplasma gondii isolation in muscle samples by mouse bioassay was higher than in PCR (P < 0.01). Results indicate that DNA from pig tissues interfered with 529-bp-PCR sensitivity, and mouse bioassay was better than PCR in detecting T. gondii in tissues from pigs. © 2006 Elsevier Inc. All rights reserved.

Stability study of fluconazole applying validated bioassay and stability-indicating LC methods

Chemical degradation of drugs may result in altered therapeutic efficacy and even toxic effects. Therefore, understanding the factors that change the stability of pharmaceuticals and identifying ways to guarantee their stability are important. In this work stability-indicating Liquid Chromatographic (LC) and bioassay methods were validated and employed in the fluconazole stability studies. The correlation of sample results from both methods was evaluated. Fluconazole raw material stability was investigated in aqueous, acid (0.1 M HCl), alkaline (0.1 M NaOH) and oxidative (3% v/v H2O2) reflux for 6 hours, by LC method. Fluconazole capsules were exposed to UVC (254 nm, 66 and 180 days), climatic chamber (40°C, 75% RH, 90 days) and oven (60°C, 60 days), these samples were analyzed by LC and bioassay methods It was found that the drug is degraded (10% decrease) with arising of a possible degradation product in an oxidative medium and UVC exposure, in all the others conditions fluconazole remained chemically stable (higher than 98%) when analyzed by LC. However when the capsules stressed samples were evaluated through bioassay very low antifungal activity was found (about 30%). Fluconazole showed to be an unstable drug and it indicates that special care must be taken during the handling, storage and quality control using appropriated methods to analyze this therapeutic agent. This work suggests monitoring the fluconazole stability by bioassay and the stability-indicating LC methods.