Study of transcripts and reactive oxygen species involved in the defence responses of Quercus suber against Phytophthora cinnamomi

The present work has the merit of exploring an insight into the activation of defence genes of Quercus suber during response to infection by Phytophthora cinnamomi. Thus, cDNA-AFLP methodology was used to identify gene fragments differentially present in the mRNA profiles of host cells of micropropagated Q. suber plantlets roots infected with zoospores of P. cinnamomi at different post challenge time points. Six candidate genes were selected based on their interesting cDNA-AFLP expression patterns and homology to genes known to play a role in defence. These six genes encode a cinnamyl alcohol dehydrogenase 2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), thaumatin-like protein (QsTLP), chitinase (QsCHI) and a 1,3-beta glucanase (QsGLU). The current work has been successful in evaluation of the expression of these genes by qRT-PCR. Data analysis revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the early hours of inoculation, while transcript profiles of thaumatin-like protein showed decreasing. No expression was detected for 1,3-beta-glucanase (QsGLU). Furthermore, the choice of suitable reference genes in any new experimental system is absolutely crucial in qRT-PCR; for this reason in this study and for the first time a set of potential reference genes were analyzed and validated for qRT-PCR normalization in the patho-system Phytophthora-Q. suber. Four candidate reference genes polimerase II (QsRPII), eukaryotic translation initiation factor 5A(QsEIF-5A), b-tubulin (QsTUB) and a medium subunit family protein of Clathrin adaptor complexes (QsCACs) were evaluated to determine the most stable internal references in Q. suber. Analysis of stability of genes was carried out using Genex software. Results indicated all these four potential reference genes assumed stable expression. Data analysis revealed that QsRPII and QsCACs were the two most stable genes, while genes QsTUB and QsEIF-5A were the third and the fourth most stable gene, respectively. In this study, a plasmid-based quantitative PCR method was developed to measure P. cinnamomi colonization during infection process of Q. suber. Plasmid-based detection of P. cinnamomi showed a gradual accumulation of the pathogen DNA in cork oak root tips up to 24 h post infection. The higher increase in P. cinnamomi/plasmid DNA ratio occurred between 18 and 24 h. One of the primary objectives of this research was to study the effect of cinnamomins (elicitins secreted by P. cinnamomin) on inducing defence mechanism against the pathogen, as recent histological and ultra-structural studies showed that P. cinnamomi was restricted to the outer cortex root fragments pre-treated with capsicien and cryptogein, suggesting that elicitins can stimulate plant defence reactions against P. cinnamomi. To complement these studies and to have a clear view of the nature of the interaction, the role of cinnamomins in the production of the oxidative burst [ROS and ROS scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)] and in the defence responses was evaluated. Cork oak seedlings were pretreated with alpha-cinnamomin and then inoculated with P. cinnamomi mycelia. Results showed a significant higher production of reactive oxygen species (ROS) (H2O2 and O2•-) in elicitin and non-elicitin treated roots in interaction with P. cinnamomi in comparison to the corresponding control. The plant group inoculated with the pathogen after cinnamomin treatment showed an earlier increase in H2O2 production but this was lower as compared with that group inoculated with P. cinnamomi alone. Also, in elicitin pre-treated group generally, a lower level of O2•− production during infection was observed as compared with inoculated roots with P. cinnamomi alone without elicitin treatment. Furthermore, in this study, we evaluated activities of antioxidant enzymes upon challenge with P. cinnamomi, with and without pretreatment with alpha cinnamomin. Results indicated that the activities of defense enzymes POD, SOD and CAT increased after P. cinnamomi inoculation when compared with those in the control group. Also, in the group treated with alpha-cinnamomin followed by P. cinnamomi inoculation, a higher level of enzymatic activities was detected as compared with elicitin non-treated group, which suggest the protective effect of alpha-cinnamomin against the pathogen due to higher elevated levels of defense enzymes POD, SOD and CAT during the infection period. Furthermore, a sensitive qPCR method was applied to measure the pathogen biomass in elicited and non-elicited Q. suber roots challenged with P. cinnamomi to elucidate the effect of cinnamomins on the colonization of P. cinnamomi. Plasmid-based quantification of P. cinnamomi showed a significant decrease in accumulation of the pathogen DNA in cork oak roots after treatment with alpha and beta-cinnamomins which attest the role of cinnamomins in promoting defense responses in cork oak against P. cinnamomi invasion.

In Vitro Ketamine CYP3A Mediated Metabolism Study using Mammalian Liver S9 Fractions, cDNA Expressed Enzymes and Liquid Chromatography Tandem Mass Spectrometry

Ketamine is widely used in medicine in combination with several benzodiazepines including midazolam. The objectives of this study were to develop a novel HPLC-MS/SRM method capable of quantifying ketamine and norketamine using an isotopic dilution strategy in biological matrices and study the formation of norketamine, the principal metabolite of ketamine with and without the presence of midazolam, a well-known CYP3A substrate. The chromatographic separation was achieved using a Thermo Betasil Phenyl 100 x 2 mm column combined with an isocratic mobile phase composed of acetonitrile, methanol, water and formic acid (60:20:20:0.4) at a flow rate of 300 μL/min. The mass spectrometer was operating in selected reaction monitoring mode and the analytical range was set at 0.05–50 μM. The precision (%CV) and accuracy (%NOM) observed were ranging from 3.9–7.8 and 95.9.2–111.1% respectively. The initial rate of formation of norketamine was determined using various ketamine concentration and Km values of 18.4 μM, 13.8 μM and 30.8 μM for rat, dog and human liver S9 fractions were observed respectively. The metabolic stability of ketamine on liver S9 fractions was significantly higher in human (T1/2 = 159.4 min) compared with rat (T1/2 = 12.6 min) and dog (T1/2 = 7.3 min) liver S9 fractions. Moreover significantly lower IC50 and Ki values observed in human compared with rat and dog liver S9 fractions. Experiments with cDNA expressed CYP3A enzymes showed the formation of norketamine is mediated by CYP3A but results suggest an important contribution from others isoenzymes, most likely CYP2C particularly in rat.

Influence of Selected Dietary Micronutrient Elements on Growth and Biochemistry of Farmed Prawns

From the present study, it is clear that all the three metals, selenium, molybdenum and cobalt have significant effect on the antioxidant status of the shrimps. Selenium and molybdenum were observed to induce peroxidative damage at elevated levels. But at the same level, cobalt did not show such an effect. Selenium was found to be growth promoting at lower levels of dietary supplementation. Even though low levels of dietary selenium had a protective effect against the lipid peroxidation, the present study indicates that high levels of dietary selenium could promote lipid peroxidation. The selenium-dependent antioxidant enzyme, GPx behaved differently in muscle and hepatopancreas. A high concentration of selenium was required for the active expression of the enzyme in the muscle, where as in hepatopancreas maximum activity was observed at lower selenium concentration. Selenium supplementation had a positive effect on GSH concentration. The other antioxidant enzymes such as GST, SOD and CAT showed enhanced activity at higher concentration of selenium. Molybdenum supplementation significantly reduced the free radical scavenger enzymes SOD and CAT. This resulted in enhanced lipid peroxidation in tissues. The activity of antioxidant enzyme GPx and the concentration of the substrate for the enzyme, GSH also were lower at elevated levels of molybdenum supplementation. In addition to this amino acids and fatty acids were also altered in molybdenum supplemented groups. In trace amounts, dietary molybdenum exerts a beneficial effect on the growth and also in the activities of the enzymes XO and SO. At the same time it also indicates a possibility of oxidative damage as a result of the peroxidation caused by the activities of the enzymes SO and XO at elevated concentrations of molybdenum is also indicated. The absorption of various trace elements was also altered by molybdenum supplementation.Among the three metals studied, cobalt was the least toxic one at the administered levels. But this metal has a significant effect on the lipid content, amino acid composition, cholesterol levels and phospholipid levels. Increased growth was also observed as a result of cobalt supplementation in shrimps. The antioxidant system of the animal was activated by dietary cobalt. Tissue levels of the trace metals were also found to be altered in cobalt supplemented groups of shrimps.These studies, thus shows that influence of dietary trace metals calls for more detailed studies in farmed shrimp. They may hold the key to growth and even disease resistance in shrimp. But this still remains as a virgin field which demands more attention, especially in view of the increasing importance of shrimp farming.

Withania somnitera and withanolide a mediated restoration of AMPA and NMDA receptor function in pilocarpine induced temporal lobe epilepsy

The onset of spontaneous seizures triggers a cascade of molecular and cellular events that eventually leads to neuronal injury and cognitive decline. The present study investigated the effect of Withania somnifera (WS) root extract and Withanolide A (WA) in restoring behavioural deficit by inhibiting oxidative stress induced alteration in glutamergic neurotransmission. The subdued performance in behavioural tests shows impaired motor coordination and memory. Histopathological investigations revealed significant neuronal loss in hippocampus of epileptic rats indicating glutamate mediated excitotoxicity. The treatment with WS and WA restored behavioural deficit and ameliorated neuronal loss. An altered redox homeostasis leading to oxidative stress is a hallmark of TLE. The antioxidant potential was afflicted in epileptic rats, evident from altered activity of SOD and CAT, down regulation of SOD and GPX expression and enhanced lipid peroxidation. The antioxidant property of WS and WA restored altered antioxidant capacity. Alteration in GDH activity and down regulation of GLAST expression resulted in enhanced glutamate content in the brain regions. The metabolism of glutamate was altered in the form of down regulated GAD expression. The alteration in synthesis, transport and metabolism resulted in further increase of the glutamate concentration at the synapse leading to glutamate mediated excitotoxicity. The decreased NMDA and AMPA receptor binding and down regulated NMDA R1, NMDA 2B and AMPA (GluR2) mRNA expression indicated altered glutamergic receptor function. The treatment with WS and WA reversed altered glutamergic receptor function, synthesis, transport and metabolism. The enhanced levels of second messenger IP3 responsible for Ca2+ mediated toxicity was reversed after treatment with WS and WA. Neurotoxics concentration of glutamate resulted in up regulation of pro apoptotic factors Bax and Caspase 8 and down regulation of anti apoptotic factor Akt resulting in neuronal death. The treatment with WS and WA resulted in activation of Akt and down regulation of Bax and caspase 8 leading to blocking of apoptotic pathway. The treatment with WS and WA resulted in reduced seizure frequency and amelioration of associated alterations suggesting the therapeutic role of Withania somnifera in temporal lobe epilepsy

Generation, description and storage of dendritic morphology data

It is generally assumed that the variability of neuronal morphology has an important effect on both the connectivity and the activity of the nervous system, but this effect has not been thoroughly investigated. Neuroanatomical archives represent a crucial tool to explore structure–function relationships in the brain. We are developing computational tools to describe, generate, store and render large sets of three–dimensional neuronal structures in a format that is compact, quantitative, accurate and readily accessible to the neuroscientist. Single–cell neuroanatomy can be characterized quantitatively at several levels. In computer–aided neuronal tracing files, a dendritic tree is described as a series of cylinders, each represented by diameter, spatial coordinates and the connectivity to other cylinders in the tree. This ‘Cartesian’ description constitutes a completely accurate mapping of dendritic morphology but it bears little intuitive information for the neuroscientist. In contrast, a classical neuroanatomical analysis characterizes neuronal dendrites on the basis of the statistical distributions of morphological parameters, e.g. maximum branching order or bifurcation asymmetry. This description is intuitively more accessible, but it only yields information on the collective anatomy of a group of dendrites, i.e. it is not complete enough to provide a precise ‘blueprint’ of the original data. We are adopting a third, intermediate level of description, which consists of the algorithmic generation of neuronal structures within a certain morphological class based on a set of ‘fundamental’, measured parameters. This description is as intuitive as a classical neuroanatomical analysis (parameters have an intuitive interpretation), and as complete as a Cartesian file (the algorithms generate and display complete neurons). The advantages of the algorithmic description of neuronal structure are immense. If an algorithm can measure the values of a handful of parameters from an experimental database and generate virtual neurons whose anatomy is statistically indistinguishable from that of their real counterparts, a great deal of data compression and amplification can be achieved. Data compression results from the quantitative and complete description of thousands of neurons with a handful of statistical distributions of parameters. Data amplification is possible because, from a set of experimental neurons, many more virtual analogues can be generated. This approach could allow one, in principle, to create and store a neuroanatomical database containing data for an entire human brain in a personal computer. We are using two programs, L–NEURON and ARBORVITAE, to investigate systematically the potential of several different algorithms for the generation of virtual neurons. Using these programs, we have generated anatomically plausible virtual neurons for several morphological classes, including guinea pig cerebellar Purkinje cells and cat spinal cord motor neurons. These virtual neurons are stored in an online electronic archive of dendritic morphology. This process highlights the potential and the limitations of the ‘computational neuroanatomy’ strategy for neuroscience databases.

Influence of UV radiation on chlorophyll, and antioxidant enzymes of wetland plants in different types of constructed wetland

A surface- and vertical subsurface-flow-constructed wetland were designed to study the response of chlorophyll and antioxidant enzymes to elevated UV radiation in three types of wetland plants (Canna indica, Phragmites austrail, and Typha augustifolia). Results showed that (1) chlorophyll content of C. indica, P. austrail, and T. augustifolia in the constructed wetland was significantly lower where UV radiation was increased by 10 and 20 % above ambient solar level than in treatment with ambient solar UV radiation (p < 0.05). (2) The malondialdehyde (MDA) content, guaiacol peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities of wetland plants increased with elevated UV radiation intensity. (3) The increased rate of MDA, SOD, POD, and CAT activities of C. indica, P. australis, and T. angustifolia by elevated UV radiation of 10 % was higher in vertical subsurface-flow-constructed wetland than in surface-flow-constructed wetland. The sensitivity of MDA, SOD, POD, and CAT activities of C. indica, P. austrail, and T. augustifolia to the elevated UV radiation was lower in surface-flow-constructed wetland than in the vertical subsurface-flow-constructed wetland, which was related to a reduction in UV radiation intensity through the dissolved organic carbon and suspended matter in the water. C. indica had the highest SOD and POD activities, which implied it is more sensitive to enhanced UV radiation. Therefore, different wetland plants had different antioxidant enzymes by elevated UV radiation, which were more sensitive in vertical subsurface-flow-constructed wetland than in surface-flow-constructed wetland.

Style and substance: some metal figurines from south-west Britain

A hoard found in Southbroom, Devizes in 1714 contained a group of copper-alloy figurines executed in both classical and local styles and depicting deities belonging to the Graeco-Roman and Gallo-Roman pantheons. The deities in a local style appear to form part of a larger tradition of figurines, predominantly found in the South-West, which are characterised both by a similar artistic style and by the use of Gallo-Roman symbolism and deities, such as the torc, ram-horned snake, carnivorous dog and Sucellus. The unique composition of the hoard in comparison with other hoards of similar date provides insights into the beliefs of Roman Britain.

The periaqueductal gray and its potential role in maternal behavior inhibition in response to predatory threats

Animals faced with conflicting cues, such as predatory threat and a given rewarding stimulus, must make rapid decisions to engage in defensive versus other appetitive behaviors. The brain mechanisms mediating such responses are poorly understood. However, the periaqueductal gray (PAG) seems particularly suitable for accomplishing this task. The PAG is thought to have, at least, two distinct general roles on the organization of motivated responses, i.e., one on the execution of defensive and reproductive behaviors, and the other on the motivational drive underlying adaptive responses. We have presently examined how the PAG would be involved in mediating the behavioral choice between mutually incompatible behaviors, such as reproduction or defense, when dams are exposed to pups and cat odor. First, we established the behavioral protocol and observed that lactating rats, simultaneously exposed to pups and cat odor, inhibited maternal behavior and expressed clear defensive responses. We have further revealed that cat odor exposure up-regulated Fos expression in the dorsal PAG, and that NMDA cytotoxic lesions therein were able to restore maternal responses, and, at the same time, block defensive responsiveness to cat odor. Potential paths mediating the dorsal PAG influences on the inhibition of appetitive (i.e., retrieving behavior) and consummatory (i.e., nursing) maternal responses are discussed. Overall, we were able to confirm the dual role of the PAG, where, in the present case, the dorsal PAG, apart from organizing defensive responses, also appears to account for the behavioral inhibition of non-defensive responses. (C) 2010 Elsevier B.V. All rights reserved.

Can therapy dogs evoke awareness of one's past and present life in persons with Alzheimer's disease?

BACKGROUND: Persons with Alzheimer's disease (AD) sometimes express themselves through behaviours that are difficult to manage for themselves and their caregivers, and to minimise these symptoms alternative methods are recommended. For some time now, animals have been introduced in different ways into the environment of persons with dementia. Animal-Assisted Therapy (AAT) includes prescribed therapy dogs visiting the person with dementia for a specific purpose. AIM: This study aims to illuminate the meaning of the lived experience of encounters with a therapy dog for persons with Alzheimer's disease. METHOD: Video recorded sessions were conducted for each visit of the dog and its handler to a person with AD (10 times/person). The observations have a life-world approach and were transcribed and analysed using a phenomenological hermeneutical approach. RESULTS: The result shows a main theme 'Being aware of one's past and present existence', meaning to connect with one's senses and memories and to reflect upon these with the dog. The time spent with the dog shows the person recounting memories and feelings, and enables an opportunity to reach the person on a cognitive level. CONCLUSIONS: The present study may contribute to health care research and provide knowledge about the use of trained therapy dogs in the care of older persons with AD in a way that might increase quality of life and well-being in persons with dementia. IMPLICATIONS FOR PRACTICE: The study might be useful for caregivers and dog handlers in the care of older persons with dementia.

INTRAOCULAR TRANSMISSIBLE VENEREAL TUMOR IN A DOG

Tumor venéreo transmissível (TVT) é uma neoplasia de células redondas que acomete a mucosa genital externa de cães, machos e fêmeas. A transmissão decorre da implantação de células tumorais durante o coito, brigas ou interações entre animais portadores e susceptíveis. Existem relatos referindo-se a localizações atípicas do TVT, mas metástases raramente ocorrem. O presente relato descreve um caso incomum de TVT, com acometimento intra-ocular e metástases nos linfonodos ingüinais, num cão Terrier Brasileiro, com seis anos de idade. O animal apresentava massas anormais de tecido no olho direito, extremidade do pênis e aumento de volume de linfonodos da região ingüinal. A histopatologia do globo ocular e as citologias da massa peniana e dos nódulos subcutâneos evidenciaram aspectos citológicos semelhantes, caracterizados por células redondas com núcleo grande e nucléolo proeminente localizado centralmente. O citoplasma apresentou-se pálido e com presença de vacúolos pequenos e arredondados. O diagnóstico de TVT com acometimento intra-ocular e metástases em linfonodos foi baseado nos achados clínicos, citológicos e histopatológicos.

Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores

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

Risk factors and presence of antibodies to Toxoplasma gondii in dogs from the coast of São Paulo State, Brazil

A toxoplasmose é causada por um protozoário parasita intracelular obrigatório, Toxoplasma gondii, e acomete vertebrados homeotérmicos incluindo animais de companhia e o homem. O cão apresenta importância epidemiológica por atuar como sentinela da infecção para o homem. O presente estudo objetivou determinar a ocorrência de anticorpos para T. gondii em 205 amostras de soro de cães do município de Ubatuba, SP, Brasil, pela reação de imunofluorescência indireta (RIFI), assim como os fatores de risco relacionados à infecção nos animais, como raça, idade, sexo, acesso a rua, ingestão de comida caseira, acesso a água não tratada e presença de roedores. 52/205 (25,4%) amostras foram positivas para toxoplasmose, com títulos variando de 16 a 256. Os resultados sorológicos apresentaram associação significativa (P<0,05) com consumo de comida caseira (45/118; 38,1%; IC95% 29,9%-47,2%) (OR=7,0; CI95% 3,0-16,6), e acesso a rua, em que aqueles que não tinham acesso a rua foram prevalentes (37/121; 30,6%; IC95% 23,1%-39,3%) (OR=0,5; CI95% 0,2-1,0). Estes resultados demonstram que a toxoplasmose na região está relacionada com problema de educação sanitária, principalmente quanto ao adequado cozimento dos alimentos, visto que a maioria dos animais positivos não apresentou associação significativa com presença de roedores ou consumo de água não tratada, porém os mesmos permaneciam em casa aos quais fora oferecida comida caseira. Portanto, a toxoplasmose consiste em um problema de saúde pública na região estudada, sendo necessárias medidas sanitárias para o controle da infecção, visto a estreita relação homem-cão e os fatores de risco presentes.

Kinematic analysis of Labrador Retrievers and Rottweilers trotting on a treadmill

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

Toxic acute hepatitis associated to the administration of prostaglandin in a dog

A prostaglandina F2a pode ser usada em caes para aumentar o volume do ejaculado em casos de inseminação artificial, criopreservação seminal ou biotecnologias de reprodução. Os efeitos colaterais após a administração da PGF2a, como taquicardia, salivação, emese, diarréia e convulsões geralmente são relacionadas com a dose utilizada. Esse trabalho objetiva relatar a ocorrência de hepatite tóxica aguda após a administração de PGF2a em um cão, e discutir a importância de se utilizar essa droga com cautela nessa espécie.

cDNA sequence and molecular modeling of a nerve growth factor from Bothrops jararacussu venomous gland

The complete nucleotide sequence of a nerve growth factor precursor from Bothrops jararacussu snake (Bj-NGF) was determined by DNA sequencing of a clone from cDNA library prepared from the poly(A) + RNA of the venom gland of B.jararacussu. cDNA encoding Bj-NGF precursor contained 723 bp in length, which encoded a prepro-NGF molecule with 241 amino acid residues. The mature Bj-NGF molecule was composed of I 18 amino acid residues with theoretical pI and molecular weight of 8.31 and 13,537, respectively. Its amino acid sequence showed 97%, 96%, 93%, 86%, 78%, 74%, 76%, 76% and 55% sequential similarities with NGFs from Crotalus durissus terrificus, Agkistrodon halys pallas, Daboia (Vipera) russelli russelli, Bungarus multicinctus, Naja sp., mouse, human, bovine and cat, respectively. Phylogenetic analyses based on the amino acid sequences of 15 NGFs separate the Elapidae family (Naja and Bungarus) from those Crotalidae snakes (Bothrops, Crotalus and Agkistrodon). The three-dimensional structure of mature Bj-NGF was modeled based on the crystal structure of the human NGF. The model reveals that the core of NGF, formed by a pair of P-sheets, is highly conserved and the major mutations are both at the three beta-hairpin loops and at the reverse turn. (C) 2002 Societe francaise de biochimie et biologic moleculaire/Editions scientifiques et medicales Elsevier SAS. All rights reserved.