Parkinson's disease relevant pathological features are manifested in male Pink1/Parkin deficient rats.

Animal disease models are important for neuroscience experimentation and in the study of neurodegenerative disorders. The major neurodegenerative disorder leading to motor impairments is Parkinson's disease (PD). The identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in PTEN-induced putative kinase 1 (Pink1) and the E3 ubiquitin ligase Parkin, two proteins involved in mitochondrial quality control, that could be harnessed to create animal models. However, to date, such models have not reproducibly recapitulated major aspects of the disease. Here, we describe the generation and phenotypic characterization of a combined Pink1/Parkin double knockout (dKO) rat, which reproducibly exhibits PD-relevant abnormalities, particularly in male animals. Motor dysfunction in Pink1/Parkin dKO rats was characterized by gait abnormalities and decreased rearing frequency, the latter of which was responsive to levodopa treatment. Pink1/Parkin dKO rats exhibited elevated plasma levels of neurofilament light chain and significant loss of tyrosine hydroxylase expression in the substantia nigra pars compacta (SNpc). Glial cell activation was also observed in the SNpc. Pink1/Parkin dKO rats showed elevated plasma and reduced cerebrospinal levels of alpha-synuclein as well as the presence of alpha-synuclein aggregates in the striatum. Further, the profile of circulating lymphocytes was altered, as elevated CD3+CD4+ T cells and reduced CD3+CD8+ T cells in Pink1/Parkin dKO rats were found. This coincided with mitochondrial dysfunction and infiltration of CD3+ T cells in the striatum. Altogether, the Pink1/Parkin dKO rats exhibited phenotypes similar to what is seen with PD patients, thus highlighting the suitability of this model for mechanistic studies of the role of Pink1 and Parkin in PD pathogenesis and as therapeutic targets.

Longitudinal in vivo metabolic labeling reveals tissue-specific mitochondrial proteome turnover rates and proteins selectively altered by parkin deficiency.

Our study utilizes a longitudinal isotopic metabolic labeling approach in vivo in combination with organelle fraction proteomics to address the role of parkin in mitochondrial protein turnover in mice. The use of metabolic labeling provides a method to quantitatively determine the global changes in protein half-lives whilst simultaneously assessing protein expression. Studying two diverse mitochondrial populations, we demonstrated the median half-life of brain striatal synaptic mitochondrial proteins is significantly greater than that of hepatic mitochondrial proteins (25.7 vs. 3.5 days). Furthermore, loss of parkin resulted in an overall, albeit modest, increase in both mitochondrial protein abundance and half-life. Pathway and functional analysis of our proteomics data identified both known and novel pathways affected by loss of parkin that are consistent with its role in both mitochondrial quality control and neurodegeneration. Our study therefore adds to a growing body of evidence suggesting dependence on parkin is low for basal mitophagy in vivo and provides a foundation for the investigation of novel parkin targets.

Adicción al tabaco y adolescencia: riesgos psicobiológicos / Psychobiological risks of tobacco during adolescence. A narrative review

Resumen La investigación moderna, tanto en humanos como preclínica, que utiliza modelos animales indica que fumar durante la edad adolescente resulta en cambios cerebrales y psicológicos a corto y largo plazo en el fumador, así como en un aumento significativo en los riesgos de desarrollar adicción al tabaco durante la vida. Por lo tanto, en la presente revisión narrativa se describirán y profundizarán los hallazgos investigativos modernos de la psicobiología de la adolescencia y los efectos del tabaco en el desarrollo, con un énfasis particular en la comprensión de los efectos psicológicos y cerebrales del consumo de tabaco durante la adolescencia, tanto a corto como a largo plazo. Se considerarán de manera detallada los avances investigativos sobre la psicobiología de la adolescencia y sus riesgos en las adicciones desde los aspectos: conductual, cognitivo, reactividad al estrés y psicobiología. Sobre esta base, se revisará la investigación sobre la psicobiología de la adolescencia y la evidencia de vulnerabilidad a la adicción durante esta etapa. Al final, se abordarán los efectos del tabaco en el cerebro y conducta durante el desarrollo adolescente y vida posterior, ya que se ha encontrado evidencia relacionada con alteraciones cerebrales crónicas en los sistemas colinérgicos y regiones cerebrales asociadas con la dependencia de la nicotina. Se espera que la revisión y divulgación de esta información en el idioma español sea de valor para la comprensión de los problemas de vulnerabilidad y predisposiciones a la adicción al tabaco en el contexto de Latinoamérica.

Abstract Tobacco use and its harmful health-related problems have become one of the largest modern preventable public health issues. Current research strongly suggests that smoking during adolescence enhances addictive smoking behaviors during life, which can be related to adolescence as a critical ontogenetic period characterized by behaviors that can increase the probability of risk-related behaviors such as sensation and novelty seeking. Adolescent development is also a period of maturation of frontal and subcortical neural systems, brain changes that underlie higher impulsivity tendencies to promote adequate learning and adaptations necessary to succeed the novel challenges of the adult life, but those changes also enhance vulnerabilities to the addictive effects of drugs. Consistent with this, tobacco use affects brain development processes which underlie long-term psychobiological alterations and the enhanced risks for tobacco addiction during adult life. Thus, the present review describes current psychobiological approaches to understand general addiction processes and tobacco addiction, highlighting the behavioral and neural short-term effects of tobacco use during adolescence and its long-term effects during adulthood. Current research has advanced on four aspects for the understanding of both the psychobiology of adolescent development and the effects of drugs of abuse during this time. The first aspect is behavioral, as adolescence is related to important changes on motivational and emotional behaviors such as sensation seeking. Other important behavioral changes are social approach, a higher variety of opportunity for personal choices, and development of personal independence. Research on a second aspect has focused on cognition. A review of research is presented showing enhanced abilities during adolescence development for reading, abstract and logical thinking, and novel problem solving. Stress reactivity is the third aspect of reviewed psychobiological mechanisms. The stress biological system undergoes important changes during adolescence, including changes on stress-related hormones and neural architecture. An important issue is that exposure to early and/or chronic stressful circumstances during adolescence could be related to higher risk to the start and maintenance of addiction states, as suggested by research assessing the disruptive effects of stress on psychobiological homeostatic processes needed to maintain stable biological and emotional regulation. The fourth aspect is psychobiology. In this section research is reviewed related to the development of monoaminergic brain circuits underlying motivation, novelty-seeking, impulsivity, and addiction processes. Using as model the previous review integration, the effects of nicotine are discussed, the essential addictive component of tobacco, on the neurochemical systems underlying tobacco addiction. Following this, important research is introduced that describes psychobiological changes during adolescence and evidence of vulnerability to addiction during this life stage. Then, current research on both short-term and long-term effects of tobacco or nicotine administration during adolescence on the brain, behavior, and cognition is introduced. The current research advances and discussions on the psychobiology of addictions in general, and tobacco addiction in particular, have been possible to a large extent from the use of animal models and preclinical research, since animal models have become crucial to identify learning, motivational, emotional, and cognitive mechanisms that underlie addictive processes, and making possible to perform experimental procedures to discover the functioning and participation of biological components. One example of such components is the cholinergic system, which is activated by nicotine and is part of the neurochemical machinery on different brain areas important for both tobacco addiction and adolescence development such as the dorsal striatum, amygdala, ventral tegmental area, nucleus accumbens, prefrontal cortex, and hippocampus. The present review and research divulgation written in Spanish are expected to clarify modern research on addiction and encourage current scientific education on the vulnerabilities and predispositions for tobacco abuse in Latin-American countries.

Agrobacterium tumefaciens mediated transformation of the aquatic carnivorous plant Utricularia gibba.

BACKGROUND: The genus Utricularia belongs to Lentibulariaceae, the largest family of carnivorous plants, which includes terrestrial, epiphytic and aquatic species. The development of specialized structures that evolved for carnivory is a feature of this genus that has been of great interest to biologists since Darwin's early studies. Utricularia gibba is itself an aquatic plant with sophisticated bladder traps having one of the most complex suction mechanisms for trapping prey. However, the molecular characterization of the mechanisms that regulate trap development and the biophysical processes involved in prey trapping are still largely unknown due to the lack of a simple and reproducible gene transfer system. RESULTS: Here, we report the establishment of a simple, fast and reproducible protocol for genetic transformation of U. gibba based on the T-DNA of Agrobacterium tumefaciens. An in vitro selection system using Phosphinotricin as a selective agent was established for U. gibba. Plant transformation was confirmed by histochemical GUS assays and PCR and qRT-PCR analyses. We report on the expression pattern of the 35S promoter and of the promoter of a trap-specific ribonuclease gene in transgenic U. gibba plants. CONCLUSIONS: The genetic transformation protocol reported here is an effective method for studying developmental biology and functional genomics of this genus of carnivorous plants and advances the utility of U. gibba as a model system to study developmental processes involved in trap formation.

Occurrence of halogenated natural products in highly consumed fish from polluted and unpolluted tropical bays in SE Brazil.

Natural compounds from the metabolism of marine organisms have been detected at high concentrations in environmental samples which are not the producers of these compounds. These natural substances are known as halogenated natural products (HNPs). HNPs are possibly toxic halogenated compounds analogous to POPs that may bioaccumulate and biomagnify along the food web and pose a further risk to human and environmental health. The present study analyzed the occurrence of HNPs in the edible muscle of the three most consumed commercial fish species in the state of Rio de Janeiro: sardine (Sardinella brasiliensis), whitemouth croaker (Micropogonias furnieri) and mullet (Mugil liza) from the highly polluted Guanabara Bay (GB) and the less polluted Ilha Grande Bay (IGB). The analytical steps included Soxhlet extraction, clean-up step and injection in a gas chromatography system coupled to a mass spectrometer operated in the electron-capture negative ion mode (GC/ECNI-MS). The compounds 2,4,6-TBP, 2,4,6-TBA, MHC-1, Q1, 6-MeO-BDE 47 and 2'-MeO-BDE 68 were found in the analyzed fish from both studied areas. Q1, 6-MeO-BDE 47 and 2'-MeO-BDE 68 showed the highest concentrations in samples. Q1 concentrations in the sardines from IGB were higher than the sardines from GB (p < 0.05) and higher than the other IGB species (p < 0.05). The differences found among the species may be related to their characteristic habitat and diet. It is noteworthy that most of these compounds do not have any toxicological reference value. Moreover, the HNPs are being detected in species of low trophic level and since this study has worked only with commercial species, these fish may be considered as a source for human exposure to these natural compounds.

Refractory Pseudomonas Bacteremia in a 2-Year-Old Sterilized by Bacteriophage Therapy.

Here, we report a complex case that involved a pediatric patient who experienced recalcitrant multidrug-resistant Pseudomonas aeruginosa infection complicated by bacteremia/sepsis; our antibacterial options were limited because of resistance, allergies, and suboptimal source control. A cocktail of 2 bacteriophages targeting the infectious organism introduced on 2 separate occasions sterilized the bacteremia.

Acurácia de informações sobre classes de medicamentos obtidas com questionário postal aplicado a idosos - Rio de Janeiro, RJ / Accuracy of drug class information obtained from a postal questionnaire to elderly respondents

OBJETIVOS: verificar a confiabilidade e a validade das informações sobre medicamentos obtidas em questionário postal, respondido por idosos, sendo a entrevista face a face o padrão-ouro. MÉTODOS: estudo seccional (Perfil de Utilização de Medicamentos por Aposentados Brasileiros), onde foram utilizadas duas abordagens (postal e domiciliar) para coleta de informações de aposentados pelo Instituto Nacional do Seguro Social (INSS) com sessenta anos de idade ou mais. Foram utilizadas também as estatísticas kappa (simples (k), ajustado (PABAK) e ponderado), índices de correlação intra-classe, indicadores de sensibilidade e especificidade, e o gráfico de Luiz et al. RESULTADOS: 234 idosos (M = 42 por cento; F = 58 por cento) responderam às duas abordagens (média = 71,7 anos). A concordância entre postal e entrevista domiciliar foi excelente (k = 0,94) para hipoglicemiantes; muito boa (k = 0,83-0,82) para inibidores da enzima conversora de angiotensina e anti-hipertensivos; boa (k = 0,71) para diuréticos; e razoável (k = 0,47) para antiinflamatórios não esteróides. A concordância foi boa (k = 0,61) para o número total de medicamentos usados. A validade da abordagem postal foi elevada, às vezes total, para os fármacos empregados no tratamento do diabetes (sensibilidade e especificidade = 100 por cento), seguidos dos anti-hipertensivos. Os menores valores obtidos foram para antiinflamatórios não esteróides (sensibilidade = 64 por cento; especificidade = 88 por cento). CONCLUSÃO: a abordagem postal pode ser usada para se obter informações acuradas sobre classes de medicamentos usados por população com idade igual ou superior a 60 anos, considerando idosos com perfil social semelhante ao dos beneficiários do INSS.

Transgenic plants of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud., from microprojectile bombardment of highly chlorophyllous embryogenic cells.

For the first time, the production of transgenic plants of the forage grass blue grama, Bouteloua gracilis [H.B.K.] Lag. ex Steud., is reported. Transgenic plants containing a gus Colon, two colons nptll fusion driven by a double CaMV35S promoter were obtained by microprojectile bombardment of the highly chlorophyllous embryogenic cell line 'TIANSJ98'. Transformed B. gracilis cell lines resisted a lethal concentration of 160 mg/l of kanamycin for at least 8 months. Chlorophyll development and growth rate were used as useful criteria for discriminating transformed from non-transformed clones. Stable integration of the transgene in the blue grama genome was demonstrated by PCR and Southern-hybridization analysis. Expression of the NPTll protein in transgenic plants grown under greenhouse conditions was confirmed indirectly by spraying kanamycin (150-250 mg/l) on plant foliage, and directly by ELISA immunological tests. Control plants sprayed with kanamycin showed foliar necrosis and reduced growth (tillering) compared to plants containing the transgene. NPTll was found in transgenic plants in levels ranging between 12.6 and 29.6 ng/mg FW of cells, as determined by ELISA reactions.

Establishment, characterization and plant regeneration from highly chlorophyllous embryogenic cell cultures of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud.

A finely dispersed, homogeneous and highly chlorophyllous cell suspension (TIANSJ98 cell line) was obtained from shoot apices of Bouteloua gracilis (H.B.K.) Lag. ex Steud. cultured on MPC medium containing MS salts supplemented with 2,4-D (1 mg/l), BAP (2 mg/l) and adenine (40 mg/l). When the TIANSJ98 cell line was grown in this medium with shaking at 180 rpm it had doubling times of 7.2 and 3.7 days in terms of fresh and dry weight, respectively. Total chlorophyll content in this cell culture ranged from 121.6 to 18.3 µg/g FW at 12 and 21 days following culture initiation. Plants regenerated from the TIANSJ98 cell line, via somatic embryogenesis, were grown to maturity and produced seeds. Although different cell culture systems have been described for cereals and grasses, to the best of our knowledge this is the first report of a highly chlorophyllous and regenerable cell suspension in Poaceae.

A prokaryotic sucrose synthase gene (susA) isolated from a filamentous nitrogen-fixing cyanobacterium encodes a protein similar to those of plants.

Sucrose synthase (SS), a key enzyme in plant carbohydrate metabolism, has recently been isolated from Anabaena sp. strain PCC 7119, and biochemically characterized; two forms (SS-I and SS-II) were detected (Porchia et al. 1999, Planta 210: 34-40). The present study describes the first isolation and characterization of a prokaryotic SS gene, susA, encoding SS-II from that strain of Anabaena. A 7 kbp DNA fragment containing an open reading frame (EMBL accession number AJ010639) with about 30-40% amino acid identity with plant SSs was isolated from an Anabaena subgenomic library. The putative SS gene was demonstrated to encode an SS protein by expression in Escherichia coli. The biochemical properties of the recombinant enzyme were identical to those of the enzyme purified from the cyanobacterial cells. The deduced amino acid sequence of the Anabaena SS diverged from every plant SS reported. The occurrence of SS in cyanobacteria of different taxonomic groups was investigated. The enzyme occurs in several filamentous nitrogen-fixing cyanobacteria but not in two species of unicellular, non-diazotrophic cyanobacteria.

Tissue-specific and developmental pattern of expression of the rice sps1 gene.

Sucrose-phosphate synthase (SPS) is one of the key regulatory enzymes in carbon assimilation and partitioning in plants. SPS plays a central role in the production of sucrose in photosynthetic cells and in the conversion of starch or fatty acids into sucrose in germinating seeds. To explore the mechanisms that regulate the tissue-specific and developmental distribution of SPS, the expression pattern of rice (Oryza sativa) sps1 (GenBank accession no. U33175) was examined by in situ reverse transcriptase-polymerase chain reaction and the expression directed by the sps1 promoter using the beta-glucuronidase reporter gene. It was found that the expression of the rice sps1 gene is limited to mesophyll cells in leaves, the scutellum of germinating seedlings, and pollen of immature inflorescences. During leaf development, the sps1 promoter directs a basipetal pattern of expression that coincides with the distribution of SPS activity during the leaf sink-to-source transition. It was also found that during the vegetative part of the growth cycle, SPS expression and enzymatic activity are highest in the youngest fully expanded leaf. Additionally, it was observed that the expression of the sps1 promoter is regulated by light and dependent on plastid development in photosynthetic tissues, whereas expression in scutellum is independent of both light and plastid development.

Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate.

Phosphorus (P) is one of the most important nutrients limiting agricultural production worldwide. In acid and alkaline soils, which make up over 70% of the world's arable land, P forms insoluble compounds that are not available for plant use. To reduce P deficiencies and ensure plant productivity, nearly 30 million tons of P fertilizer are applied every year. Up to 80% of the applied P fertilizer is lost because it becomes immobile and unavailable for plant uptake. Therefore, the development of novel plant varieties more efficient in the use of P represents the best alternative to reduce the use of P fertilizers and achieve a more sustainable agriculture. We show here that the ability to use insoluble P compounds can be significantly enhanced by engineering plants to produce more organic acids. Our results show that when compared to the controls, citrate-overproducing plants yield more leaf and fruit biomass when grown under P-limiting conditions and require less P fertilizer to achieve optimal growth.

Organic acid metabolism in plants: from adaptive physiology to transgenic varieties for cultivation in extreme soils.

During the last 20 years increasing experimental evidence has associated organic acid metabolism with plant tolerance to environmental stress. Current knowledge shows that organic acids not only act as intermediates in carbon metabolism but also as key components in mechanisms that some plants use to cope with nutrient deficiencies, metal tolerance and plant-microbe interactions operating at the root-soil interphase. In this review we summarize recent knowledge on the physiology and occurrence of organic acids in plants and their special relevance concerning nitrate reduction, phosphorus and iron acquisition, aluminum tolerance and soil ecology. We also discuss novel findings in relation to the biotechnological manipulation of organic acids in transgenic models ranging from cell cultures to whole plants. This novel perspective of organic acid metabolism and its potential manipulation may represent a way to understand fundamental aspects of plant physiology and lead to new strategies to obtain crop varieties better adapted to environmental and mineral stress.

Completion of the sexual cycle and demonstration of genetic recombination in Ustilago maydis in vitro.

The heterobasidiomycetes responsible for plant smuts obligatorily require their hosts for the completion of the sexual cycle. Accordingly, the sexual cycle of these fungi could so far be studied only by infecting host plants. We have now induced Ustilago maydis, the causative agent of corn smut, to traverse the whole life cycle by growing mixtures of mating-compatible strains of the fungus on a porous membrane placed on top of embryogenic cell cultures of its host Zea mays. Under these conditions, mating, karyogamy and meiosis take place, and the fungus induces differentiation of the plant cells. These results suggest that embryogenic maize cells produce diffusible compounds needed for completion of the sexual cycle of U. maydis, as the plant does for the pathogen during infection.

Transgenic plants for tropical regions: some considerations about their development and their transfer to the small farmer.

Biotechnological applications, especially transgenic plants, probably hold the most promise in augmenting agricultural production in the first decades of the next millennium. However, the application of these technologies to the agriculture of tropical regions where the largest areas of low productivity are located, and where they are most needed, remains a major challenge. In this paper, some of the important issues that need to be considered to ensure that plant biotechnology is effectively transferred to the developing world are discussed.

Identification of a sequence element involved in AC2-mediated transactivation of the pepper huasteco virus coat protein gene.

The geminivirus AC2 gene product transactivates the expression of the coat and movement protein (CP and BV1) genes, and this effect seems to be mediated by specific although hitherto unknown cis-acting elements. In this work we examined regions from the CP and BV1 gene promoters of pepper huasteco virus (PHV) to define the sequence elements involved in regulation by AC2. Results from transient gene expression and transgenic plant assays suggest that a truncated 115-nt CP promoter is still responsive to the viral transactivator. This promoter contains three elements similar to a sequence motif termed conserved late element (CLE), which is found in the regulatory regions of many geminiviruses and that was previously suggested, on a theoretical basis, to be a potential functional target for AC2 (Argüello-Astorga et al. (1994), Virology 203, 90-100). To confirm these results, an oligonucleotide containing two CLE motifs was synthesized and characterized in gain-of-function experiments. Transient expression assays showed that this 29-nt sequence is able to confer AC2 responsiveness to heterologous promoters. A smaller oligonucleotide (16 nt) containing a single CLE also conferred this activity. In addition, when the CLE motifs were mutated in their original context (truncated 115-nt promoter), this modified promoter lost its ability to be transactivated by AC2. All these results support the involvement, at least in the case of PHV, of CLE sequences in the process of transactivation.