Matched case-control study evaluating the frequency of the main agents associated with neonatal diarrhea in piglets

A case-control study was carried out in litters of 1 to 7-day-old piglets to identify the main infectious agents involved with neonatal diarrhea in pigs. Fecal samples (n=276) from piglets were collected on pig farms in the State of Rio Grande do Sul, Brazil, from May to September 2007. Litters with diarrhea were considered cases (n=129) and normal litters (n=147) controls. The samples were examined by latex agglutination test, PAGE, conventional isolating techniques, ELISA, PCR, and microscopic methods in order to detect rotavirus, bacterial pathogens (Escherichia coli, Clostridium perfringens type A and C, and Clostridium difficile), and parasites (Coccidian and Cryptosporidium spp.). Outbreaks of diarrhea were not observed during sampling. At least one agent was detected in fecal samples on 25 out of 28 farms (89.3%) and in 16 farms (57.1%) more than one agent was found. The main agents diagnosed were Coccidia (42.86%) and rotavirus (39.29%). The main agents identified in litters with diarrhea were Clostridium difficile (10.6%), Clostridium perfringens type A (8.8%) and rotavirus (7.5%); in control litters, Clostridium difficile (16.6%) and Coccidian (8.5%). Beta hemolytic Escherichia coli and Clostridium perfringens type C were not detected. When compared with controls, no agent was significantly associated with diarrhea in case litters. These findings stress the need for caution in the interpretation of laboratorial diagnosis of mild diarrhea in neonatal pigs, as the sole detection of an agent does not necessarily indicate that it is the cause of the problem.

Agents that affect cAMP levels or protein kinase A activity modulate memory consolidation when injected into rat hippocampus but not amygdala

Male Wistar rats were trained in one-trial step-down inhibitory avoidance using a 0.4-mA footshock. At various times after training (0, 1.5, 3, 6 and 9 h for the animals implanted into the CA1 region of the hippocampus; 0 and 3 h for those implanted into the amygdala), these animals received microinfusions of SKF38393 (7.5 µg/side), SCH23390 (0.5 µg/side), norepinephrine (0.3 µg/side), timolol (0.3 µg/side), 8-OH-DPAT (2.5 µg/side), NAN-190 (2.5 µg/side), forskolin (0.5 µg/side), KT5720 (0.5 µg/side) or 8-Br-cAMP (1.25 µg/side). Rats were tested for retention 24 h after training. When given into the hippocampus 0 h post-training, norepinephrine enhanced memory whereas KT5720 was amnestic. When given 1.5 h after training, all treatments were ineffective. When given 3 or 6 h post-training, 8-Br-cAMP, forskolin, SKF38393, norepinephrine and NAN-190 caused memory facilitation, while KT5720, SCH23390, timolol and 8-OH-DPAT caused retrograde amnesia. Again, at 9 h after training, all treatments were ineffective. When given into the amygdala, norepinephrine caused retrograde facilitation at 0 h after training. The other drugs infused into the amygdala did not cause any significant effect. These data suggest that in the hippocampus, but not in the amygdala, a cAMP/protein kinase A pathway is involved in memory consolidation at 3 and 6 h after training, which is regulated by D1, ß, and 5HT1A receptors. This correlates with data on increased post-training cAMP levels and a dual peak of protein kinase A activity and CREB-P levels (at 0 and 3-6 h) in rat hippocampus after training in this task. These results suggest that the hippocampus, but not the amygdala, is involved in long-term storage of step-down inhibitory avoidance in the rat.

Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents)

This review highlights the current advances in knowledge about the safety, efficacy, quality control, marketing and regulatory aspects of botanical medicines. Phytotherapeutic agents are standardized herbal preparations consisting of complex mixtures of one or more plants which contain as active ingredients plant parts or plant material in the crude or processed state. A marked growth in the worldwide phytotherapeutic market has occurred over the last 15 years. For the European and USA markets alone, this will reach about $7 billion and $5 billion per annum, respectively, in 1999, and has thus attracted the interest of most large pharmaceutical companies. Insufficient data exist for most plants to guarantee their quality, efficacy and safety. The idea that herbal drugs are safe and free from side effects is false. Plants contain hundreds of constituents and some of them are very toxic, such as the most cytotoxic anti-cancer plant-derived drugs, digitalis and the pyrrolizidine alkaloids, etc. However, the adverse effects of phytotherapeutic agents are less frequent compared with synthetic drugs, but well-controlled clinical trials have now confirmed that such effects really exist. Several regulatory models for herbal medicines are currently available including prescription drugs, over-the-counter substances, traditional medicines and dietary supplements. Harmonization and improvement in the processes of regulation is needed, and the general tendency is to perpetuate the German Commission E experience, which combines scientific studies and traditional knowledge (monographs). Finally, the trend in the domestication, production and biotechnological studies and genetic improvement of medicinal plants, instead of the use of plants harvested in the wild, will offer great advantages, since it will be possible to obtain uniform and high quality raw materials which are fundamental to the efficacy and safety of herbal drugs.

Dopaminergic profile of new heterocyclic N-phenylpiperazine derivatives

Dopamine constitutes about 80% of the content of central catecholamines and has a crucial role in the etiology of several neuropsychiatric disorders, including Parkinson's disease, depression and schizophrenia. Several dopaminergic drugs are used to treat these pathologies, but many problems are attributed to these therapies. Within this context, the search for new more efficient dopaminergic agents with less adverse effects represents a vast research field. The aim of the present study was to report the structural design of two N-phenylpiperazine derivatives, compound 4: 1-[1-(4-chlorophenyl)-1H-4-pyrazolylmethyl]-4-phenylhexahydropyrazine and compound 5: 1-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-ylmethyl]-4-phenylhexahydropyrazine, planned to be dopamine ligands, and their dopaminergic action profile. The two compounds were assayed (dose range of 15-40 mg/kg) in three experimental models: 1) blockade of amphetamine (30 mg/kg, ip)-induced stereotypy in rats; 2) the catalepsy test in mice, and 3) apomorphine (1 mg/kg, ip)-induced hypothermia in mice. Both derivatives induced cataleptic behavior (40 mg/kg, ip) and a hypothermic response (30 mg/kg, ip) which was not prevented by haloperidol (0.5 mg/kg, ip). Compound 5 (30 mg/kg, ip) also presented a synergistic hypothermic effect with apomorphine (1 mg/kg, ip). Only compound 4 (30 mg/kg, ip) significantly blocked the amphetamine-induced stereotypy in rats. The N-phenylpiperazine derivatives 4 and 5 seem to have a peculiar profile of action on dopaminergic functions. On the basis of the results of catalepsy and amphetamine-induced stereotypy, the compounds demonstrated an inhibitory effect on dopaminergic behaviors. However, their hypothermic effect is compatible with the stimulation of dopaminergic function which seems not to be mediated by D2/D3 receptors.

Antiretroviral agents and acid-base balance at delivery of the neonate

Limited evidence is available regarding antiretroviral (ARV) safety for uninfected infants exposed to these drugs in utero. Our objective was to determine if ARV administered to pregnant women is associated with decreasing umbilical arterial pH and base excess in uninfected infants. A prospective study was conducted on 57 neonates divided into three groups: ZDV group, born to mothers taking zidovudine (N = 20), triple therapy (TT) group, born to mothers taking zidovudine + lamivudine + nelfinavir (N = 25), and control group (N = 12), born to uninfected mothers. Umbilical cord blood was used to determine umbilical artery gases. A test was performed to calculate the sample by comparing means by the unpaired one-tailed t-test, with a = 0.05 and ß = 20%, indicating the need for a sample of 18 newborn infants for the study groups to detect differences higher than 20%. The control and ARV groups were similar in gestational age, birth weight, and Apgar scores. Values of pH, pCO2, bicarbonate, and base excess in cord arterial blood obtained at delivery from the newborns exposed to TT were 7.23, 43.2 mmHg, 19.5 mEq/L, and -8.5 nmol/L, respectively, with no significant difference compared to the control and ZDV groups. We conclude that intrauterine exposure to ARV is not associated with a pathological decrease in umbilical arterial pH or base excess. While our data are reassuring, follow-up is still limited and needs to be continued into adulthood because of the possible potential for adverse effects of triple antiretroviral agents.

An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats

We investigated whether liver injury by dual exposure to ethanol and carbon tetrachloride (EtOH + CCl4) for 15 weeks would persist after hepatotoxic agents were removed (EtOH + CCl4/8wR). After 15 weeks of hepatic injury with ethanol (5.5%, m/v) and carbon tetrachloride (0.05, mL/kg, ip), 5 of 11 female Wistar rats were sacrificed. The other 6 rats were maintained for an additional 8 weeks without hepatotoxic agents. Ultrasonography showed increased liver echogenicity and dilation of portal vein caliber in both groups (EtOH + CCl4: 0.22 ± 0.01 cm, P < 0.001; EtOH + CCl4/8wR: 0.21 ± 0.02 cm, P < 0.01) vs control (0.16 ± 0.02 cm). Histopathology showed regenerative nodules in both experimental groups. Histomorphometry revealed increased fibrosis content in both groups (EtOH + CCl4: 12.6 ± 2.64%, P < 0.001; EtOH + CCl4/8wR: 10.4 ± 1.36%, P < 0.05) vs control (2.2 ± 1.21%). Collagen types I and III were increased in groups EtOH + CCl4 (collagen I: 2.5 ± 1.3%, P < 0.01; collagen III: 1.3 ± 0.2%, P < 0.05) and EtOH + CCl4/8wR (collagen I: 1.8 ± 0.06%, P < 0.05; collagen III: 1.5 ± 0.8%, P < 0.01) vs control (collagen I: 0.38 ± 0.11%; collagen III: 0.25 ± 0.06%). Tissue transglutaminase increased in both groups (EtOH + CCl4: 66.4 ± 8%, P < 0.01; EtOH + CCl4/8wR: 58.8 ± 21%, P < 0.01) vs control (7.9 ± 0.8%). Cirrhosis caused by the association of CCl4-EtOH remained for at least 8 weeks after removal of these hepatotoxic agents. Ultrasound images can be a useful tool to evaluate advanced hepatic alterations.

Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas - FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system

Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/β-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator β-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/β-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X β-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3β inhibitor (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80% of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38% of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64%, which are the dominant β-catenin signaling cells and decreased β-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/β-catenin signaling inhibitor NCTD.

Prevention of enzymatic browning of yacon flour by the combined use of anti-browning agents and the study of its chemical composition

Yacon roots present functional properties because of the high levels of fructooligosaccharides (FOS), which are considered as prebiotic fibers. In addition, yacon roots are rich in phenolic compounds. During the processing of yacon, the freshly cut surface undergoes rapid enzymatic browning. Control of enzymatic browning during processing is very important to preserve the appearance of yacon flour. In this study, it was evaluated the combined effect of anti-browning agents (ascorbic acid, citric acid and L-cysteine) on the inhibition of enzymatic browning of yacon, using Response Surface Methodology. The yacon pre-treated with anti-browning agents in concentrations of 15.0 mM for ascorbic acid, 7.5 mM for citric acid and 10.0 mM for L-cysteine was used for the processing of flour. Yacon flour presented an attractive color and good sensory properties, without residual aroma. The contents of FOS and phenolic compounds obtained in yacon flour were 28.60 g.100 g- 1 and 1.35 g.100 g- 1. Yacon flour can be considered as a potential functional food, especially due to high levels of FOS, which allows for its use in formulation of various foods.

Sementes de ipê-branco (Tabebuia roseo-alba (Ridl.) Sand. - Bignoniaceae): temperatura e substrato para o teste de germinação

A pesquisa avaliou a temperatura e o substrato para o teste de germinação das sementes de ipê-branco (Tabebuia roseo-alba (Ridl.) Sand.). Em mesa termogradiente, foram avaliados 11 intervalos de temperatura entre 15°C e 35°C, sob oito horas de fotoperíodo diário. Foram analisados também os substratos papel (sobre papel e rolo de papel) e vermiculita (entre vermiculita) sob 30°C e 35°C, em germinadores com oito horas de fotoperíodo diário. As sementes e as plântulas foram avaliadas diariamente, considerando os critérios indicados nas regras para análise de sementes, e calculados a porcentagem e o índice de velocidade de germinação. A temperatura e o substrato interferem na germinação das sementes de ipê-branco; a condição mais favorável para o teste de germinação dessas sementes é 30°C em substrato papel.

Armazenamento de sementes de ipê-branco e ipê-roxo em diferentes embalagens e ambientes

O armazenamento em condições ideais é de fundamental importância para a conservação da qualidade fisiológica de um lote de sementes. Nesse trabalho procurou-se identificar a condição adequada para o armazenamento de sementes de ipê-branco (Tabebuia roseo-alba (Ridl.) Sand.) e de ipê-roxo (Tabebuia impetiginosa (Mart.) Standl.), bem como avaliar métodos para estimar alterações na viabilidade e no vigor dessas sementes. Diferentes grupos de sementes foram acondicionados em saco de polietileno, saco de papel Kraft e lata e armazenados por até 300 dias em condições de laboratório, geladeira e câmara refrigerada. No início do armazenamento e a cada 60 dias, determinaram-se o teor de água, a porcentagem e a velocidade de germinação e a condutividade elétrica da solução de embebição das sementes. A melhor condição para conservação de sementes de T. roseo-alba e T. impetiginosa foi obtida com o acondicionamento em lata e manutenção em geladeira. Sementes de T. impetiginosa também podem ser conservadas embaladas em saco de polietileno, saco de papel ou lata quando estocadas em câmara refrigerada. Alterações no vigor de sementes dessas espécies são primeiramente identificadas pela redução da velocidade de germinação.

Conservação de sementes de ipê-roxo (Tabebuia impetiginosa (Mart. ex DC.) Standl.) em nitrogênio líquido

O ipê-roxo (Tabebuia impetiginosa (Mart. ex DC.) Standl.) é espécie de alto valor econômico, ornamental e medicinal e está na relação das espécies para conservação genética ex situ, no Instituto Florestal de São Paulo. O objetivo deste trabalho foi o de estudar o comportamento fisiológico de sementes de ipê-roxo, conservadas com diferentes teores de água em nitrogênio líquido, por 360 dias. Frutos maduros foram colocados sobre bancada de alvenaria, em interior de laboratório e sob luz indireta, para posterior extração manual das sementes. Primeiramente, foi determinado o grau de umidade do lote e obtida a amostra do tratamento controle, com o maior teor de água a ser estudado (18,3%). Em seguida, as sementes remanescentes foram submetidas à secagem, em secador com circulação de ar a 30ºC, para a obtenção dos demais tratamentos (12,5, 8,4 e 4,2% de água). Amostras de sementes de cada um dos teores de água foram armazenadas em nitrogênio líquido (-196ºC), sendo submetidas às avaliações fisiológicas no início e após 120, 240 e 360 dias de armazenamento. O delineamento experimental foi o inteiramente casualizado com quatro tratamentos (graus de umidade), avaliados separadamente para cada época de armazenamento. A comparação das médias foi realizada pelo teste Tukey, a 5% de probabilidade. As sementes de ipê-roxo podem ser previamente desidratadas a baixo teor de água (4,2%) e, posteriormente, conservadas em nitrogênio líquido por, pelo menos, 360 dias.

Conservação de sementes de ipê-amarelo (Tabebuia chrysotricha (Mart. ex A. DC.) standl.) em função do teor de água das sementes e da temperatura do armazenamento

O ipê-amarelo (Tabebuia chrysotricha (Mart. ex A. DC.) Standl.) é uma espécie arbórea de alto valor ecológico, econômico e paisagístico. Suas sementes apresentam variação significativa na qualidade durante o armazenamento, o que representa dificuldades no desenvolvimento de técnicas de propagação. O objetivo desta pesquisa foi estudar o comportamento fisiológico das sementes de ipê-amarelo durante o armazenamento através de variações no teor de água das sementes e na temperatura do ambiente. Frutos colhidos manualmente de plantas matrizes foram colocados para secagem e posterior extração das sementes. Primeiramente, foi determinado o teor de água inicial do lote e, paralelamente, obtida a porção com o maior teor de água a ser estudado (21,1%). Em seguida, as sementes remanescentes foram submetidas à secagem, em secador com circulação forçada de ar a 28 ± 2ºC, por diferentes períodos, obtendo-se teores de água de 15,9, 13,6, 11,9 e 8,5%. As porções correspondentes aos diferentes teores de água foram armazenadas a -12, 10 e 20ºC. No início e a cada 30 dias até 270 dias de armazenamento, as sementes foram submetidas às avaliações de teor de água, germinação, emergência, velocidade de emergência e comprimento da parte aérea. O mais alto e o mais baixo teor de água estudado (21,1% e 8,5%) são prejudiciais à qualidade fisiológica das sementes mesmo à temperatura extremamente baixa (-12ºC) de armazenamento. A conservação das sementes de ipê-amarelo (Tabebuia chrysotricha) é favorecida quando armazenadas com teor de água em torno de 11,9% nas temperaturas de 10 e -12ºC.

Teste de raios X para avaliação do potencial fisiológico de sementes de ipê-roxo

Em espécies multiplicadas comercialmente via semeadura indireta, como várias essências florestais, a utilização de sementes de alto potencial fisiológico é imprescindível para garantir o desenvolvimento e estabelecimento adequado das plantas no campo. Assim, diversas técnicas têm sido utilizadas para avaliar a integridade das sementes e, dentre elas, o teste de raios X. O objetivo no trabalho foi avaliar o potencial fisiológico das sementes de ipê roxo (Tabebuia heptaphylla), após separação por cor e tamanho, por meio dos testes de raios X e de germinação. As sementes foram classificadas pela cor, utilizando carta de cor de Munsell, e tamanho, utilizando peneira de crivos oblongos, obtendo-se materiais das frações amarelo-claro maiores, amarelo-claro menores, amarelo-escuras maiores, amarelo-escuras menores, mistura de amarelo-claro maiores, amarelo-claro menores e amarelo-escuro maiores e testemunha (sem classificação). Em seguida, as sementes das diferentes frações, foram submetidas a avaliações da integridade física e viabilidade, por meio de teste de raios X e de germinação, respectivamente. Adotou-se delineamento experimental inteiramente casualizado, com seis tratamentos e cinco repetições, comparando-se as médias pelo teste de Scott-Knott (p < 0,05). O teste de raios X é eficiente na avaliação da morfologia interna de sementes de ipê-roxo e sua relação com o potencial fisiológico. A coloração e o tamanho das sementes de ipê-roxo influenciam o potencial fisiológico das mesmas.

Accelerated aging of ipê seeds under controlled conditions of storage

This research was aimed at studying effects of storage and accelerated aging on germination and profile of storage proteins in Handroanthus albus seeds. These were stored into a cold chamber (± 8 ºC; RH ± 40%) and after periods of 0, 3, 6, 9, and 12 months of storage, were subjected to accelerated aging for 0, 24, 48, 72, and 96 hours. Relationships between germination and proteins profile were assessed. Germination test was performed at 25 ºC, under constant light. For protein extraction, 125 mg of seeds were macerated in 2 mL of extraction buffer (1M Tris-HCl; pH 8.8) and applied to SDS-PAGE polyacrylamide gel at 80 V .15 h-1. Twelve month storage, combined with 72 hours accelerated aging have increased germination in approximately 65% when compared to non-aged seeds or to seeds with 24 h of accelerated aging. Besides beneficial effects, degradation and synthesis of different proteins were observed. It was concluded that germination of Handroanthus albus seeds, when not subjected to accelerated aging, is favored by storage in cold chamber during three to six months, or from nine to 12 months when subjected to accelerated aging process. Storage proteins may be associated to those increases, and hence further studies are needed.