Increased dopamine turnover in the prefrontal cortex impairs spatial working memory performance in rats and monkeys.

The selective activation of the prefrontal cortical dopamine system by mild stress can be mimicked by anxiogenic beta-carbolines such as FG7142. To investigate the functional relevance of elevated levels of dopamine turnover in the prefrontal cortex, the current study examined the effects of FG7142 on the performance of spatial working memory tasks in the rat and monkey. FG7142 selectively increased prefrontal cortical dopamine turnover in rats and significantly impaired performance on spatial working memory tasks in both rats and monkeys. Spatial discrimination, a task with similar motor and motivational demands (rats), or delayed response performance following zero-second delays (monkeys) was unaffected by FG7142. Further, biochemical analysis in rats revealed a significant positive correlation between dopamine turnover in the prefrontal cortex and cognitive impairment on the delayed alternation task. The cognitive deficits in both rats and monkeys were prevented by pretreatment with the benzodiazepine receptor antagonist, RO15-1788, which blocked the increase in dopamine turnover and by the dopamine receptor antagonists, haloperidol, clozapine, and SCH23390. These findings indicate that excessive dopamine activity in the prefrontal cortex is detrimental to cognitive functions mediated by the prefrontal cortex.

exl protein specifically binds BLE1, a bicoid mRNA localization element, and is required for one phase of its activity.

Localization of mRNAs, a crucial step in the early development of some animals, has been shown to be directed by cis-acting elements that presumably interact with localization factors. Here we identify a protein, exl, that binds to BLE1, an RNA localization element from the Drosophila bicoid mRNA. Using mutations in BLE1, we demonstrate a correlation between in vitro exl binding and one phase of in vivo localization directed by BLE1, implicating exl in that localization event. Furthermore, the same phase of localization is disrupted in exuperantia mutants, suggesting that exl and exuperantia proteins interact. Identification of a protein that binds specifically to an mRNA localization element and acts in mRNA localization opens the way for a biochemical analysis of this process.

Efeitos de dois modelos experimentais de dietas hipercalóricas sobre parâmetros comportamentais e biológicos em ratos da linhagem Wistar

O objetivo deste estudo foi investigar os efeitos de dois modelos experimentais de dietas hipercalóricas em comportamentos de ansiedade, processos de aprendizagem e memória e alterações metabólicas. Os animais foram divididos em seis grupos experimentais, de acordo com a condição nutricional. 1) Controle (C); 2) Dieta de Cafeteria (DC); 3) Dieta Hiperlipídica (DH); 4) Controle AIN-93 (C/AIN-93); 5) Dieta de Cafeteria AIN-93 (DC/AIN-93), e 6) Dieta Hiperlipídica AIN-93 (DH/AIN-93). Posteriormente, os grupos foram subdivididos em dois grupos independentes, conforme a tarefa à qual foram submetidos. Pesagens foram realizadas semanalmente até os 98 dias de vida; foram verificados os pesos do fígado, do coração e o peso de tecido adiposo retroperitoneal e epididimal e foram realizadas dosagens de glicose, triglicérides, TGO e TGP no soro e gordura total, colesterol total e triglicérides no fígado. Os testes utilizados: Labirinto em T Elevado (LTE), Caixa Claro/Escuro e Labirinto Aquático de Morris (LAM). Os resultados de peso corporal, os dados comportamentais do LAM, do LTE e os dados de peso dos tecidos extraídos no dia do sacrifício e as análises bioquímicas foram submetidos a uma Análise de Variância (ANOVA). Quando apropriado, foi utilizado o teste de comparações múltiplas de Newman-Keuls (p< 0,05). Os dados comportamentais do teste claro/escuro foram submetidos ao teste t-Student (p< 0,05). Animais tratados com dieta hiperlipídica apresentaram maiores medidas de peso e ganho de peso comparados aos animais controle e dieta de cafeteria, tratados com pellet e com dieta AIN-93. Animais DH1, DC1, DH1 AIN-93, DH2 AIN-93 e DH2 apresentaram maior peso no dia do sacrifício. Animais DH1, DH1 AIN-93, DH2 e DH2 AIN-93 apresentaram maior acúmulo dos tecidos adiposos retroperitoneal e epididimal. Animais DH1 AIN-93 e DC2 AIN-93 apresentaram maiores níveis de glicose. Animais C2, DH2 e DC2 apresentaram maiores níveis de triglicérides. Animais DH1 e C1 apresentaram menores valores de TGO. Animais C2 e C2 AIN-93 apresentaram maiores níveis de TGO. Animais C1, DH1, C2 e DH2 apresentaram maiores níveis de TGP. Animais DH1 AIN-93, DH1, DH2 e DH2 AIN-93 apresentaram maiores valores de gordura total no fígado. Animais DH1 AIN-93 e DH2 apresentaram maiores níveis de colesterol no fígado. Animais DH1, DC1, DH2 e DH2 AIN-93 apresentaram maiores níveis de triglicérides no fígado. Com relação ao consumo alimentar, animais DH apresentaram maior consumo calórico e maior consumo lipídico quando comparados aos animais C e DC, com ração em pellet ou dieta AIN-93. Com relação ao LTE, não foram verificadas diferenças nas esquivas e na fuga. Animais DC1, DH1 e DH1 AIN-93 apresentaram menores níveis de ansiedade verificados a partir dos dados do teste da caixa claro-escuro. Animais DC2 AIN-93 apresentaram pior desempenho em tarefa de memória. Os dados obtidos a partir deste estudo demonstraram que as dietas utilizadas foram capazes de acarretar ganho de peso, acúmulo de tecido adiposo, alterações metabólicas, diminuição da ansiedade nos animais e pior desempenho em uma tarefa de memória em um dos grupos nutricionais.

Impaired renal endothelial nitric oxide synthase and reticulocyte production as modulators of hypertension induced by recombinant human erythropoietin in the rat

INTRODUCTION AND AIMS: Hypertension is a common side effect of recombinant human erythropoietin (rHuEPO) therapy; however, the exact pathways remain to be elucidated. The discovery of non-hematopoietic actions of rHuEPO increased the number of patients that could putatively benefit from this therapy; however, to achieve those effects higher doses are usually needed, which increase the risk and incidence of adverse events. Our aim was to study the effect of a broad range of rHuEPO doses on hematological and biochemical parameters, blood pressure and renal function and damage in the rat, focusing on endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factors (HIFs). METHODS: Male Wistar rats were divided in 5 groups receiving different doses of rHuEPO (100, 200, 400 and 600 IU/kg body weight (BW)/week) and saline solution (control), during 3 weeks. Blood and 24h urine were collected to perform hematological and biochemical analysis. Blood pressure (BP) was measured by the tail-cuff method. The kidney tissue was collected to mRNA and protein expression assays and to characterize renal lesions. RESULTS: A dose-dependent increase in red blood cells count, hematocrit and hemoglobin levels was found with rHuEPO therapy, in rHuEPO200, rHuEPO400 and rHuEPO600 groups. Increased reticulocyte count was found in the rHuEPO400 and rHuEPO600 groups. BP raised in all groups receiving rHuEPO. The rHuEPO200 and rHuEPO600 groups presented increased kidney protein levels of HIF2α and a reduction in kidney protein levels of eNOS, along with the highest grade of vascular and tubular renal lesions. CONCLUSIONS: Our study showed that rHuEPO-induced hypertension might involve indirect (hematological) and direct (renal) effects which varies according to the dose used. Thus, rHuEPO therapy should be performed rationally and under adequate surveillance, as hypertension develops even with lower doses. Especial caution with higher doses should be taken, as rHuEPO-induced hypertension leads to early renal damage without alterations in traditional markers of renal function, thus masking the serious adverse effects and risks.

Genetically engineered silk and genipin-enhanced fibrin hydrogel for annulus fibrosus repair

INTRODUCTION: Around 80% of people are affected by low back pain at least once in their life, often caused by trauma provoking intervertebral disc (IVD) herniation and/or IVD degeneration. Apart from some promising approaches for nucleus pulposus repair, so far no treatment or repair is available for the outer fibrous tissue, annulus fibrosus (AF). We aimed for sealing and repairing an AF injury in a bovine IVD organ culture model in vitro over 14 days under different loading conditions. For this purpose, a silk fleece composite from Bombyx mori silk was combined with genipin-enhanced fibrin hydrogel [1]. METHODS: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue, followed by cutting out the IVDs [2]. Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described. On the next day, injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35- 55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d. Complex loading was applied by a custom built 2 degree of freedom bioreactor [3]. After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy-proline) were determined. Finally, real-time qPCR of major IVD marker genes was performed. RESULTS: The silk seal closing the injury site could successfully withstand the forces of all three loading conditions with no misplacement over the two weeks’ culture. Nevertheless, disc height of the repaired discs did not significantly differ from the injured group. The disc phenotype could be maintained as demonstrated by biochemical analysis of gene expression, cell activity, DNA-, collagen- and GAG content. The silk itself was evaluated to be highly biocompatible for hMSC, as revealed by cytotoxicity assays. DISCUSSION & CONCLUSIONS: The silk can be considered a highly-elastic and biocompatible material for AF closure and the genipin-enhanced fibrin hydrogel has also good biomechanical properties. However, the cyto-compatibility of genipin seems rather poor and other hydrogels and/or cross-linkers should be looked into. REFERENCES: 1 C.C. Guterl et al. (2014) Characterization of Mechanics and Cytocompatibility of Fibrin Genipin Annulus Fibrosus Sealant with the Addition of Cell Adhesion Molecules, Tissue Eng Part A 2 S.C. Chan, B. Gantenbein-Ritter (2012) Preparation of intact bovine tail intervertebral discs for organ culture, J Vis Exp 3 B Gantenbein et al. (2015) Organ Culture Bioreactors - Platforms to Study Human Intervertebral Disc Degeneration and Regenerative Therapy, Curr Stem Cell Res Ther [epub ahead of print] ACKNOWLEDGEMENTS: This project is supported by the Gebert Rüf Stiftung project # GRS-028/13.

Chlorophyll catabolism and gene expression in the peel of ripening banana fruits

The biochemical and molecular basis of chlorophyll (Chl) catabolism in bananas was investigated during ripening at 20°C and at an elevated temperature (35°C) where degreening is inhibited. Biochemical analysis showed that Chl breakdown products could be isolated from fruit ripened at both temperatures. The coloured breakdown products, chlorophyllide and pheophorbide, were not detected at any stage of ripening in the two treatments; however, a non-fluorescent Chl catabolite accumulated to a higher concentration at 20 than at 35°C. To investigate the ripening-related gene expression associated with these changes, a cDNA library was generated from the peel of fruit ripened at 20°C. Differential screening of this library produced 20 non-redundant families of clones including those encoding enzymes involved in ethylene biosynthesis, respiration, starch metabolism, cell wall degradation and other metabolic events. The expression of these genes was followed by northern analysis in fruit ripened at 20 and 35°C.

Treatment of eight dogs with nasal tumours with a alternating doses of doxorubicin and carboplatin in conjunction with oral piroxicam

Objective To determine the efficacy and toxicity of chemotherapy in the treatment of canine nasal tumours. Design Retrospective clinical study Procedure Eight dogs with histologically confirmed nasal tumours were staged by means of complete blood count, serum biochemical analysis, cytological analysis of fine needle aspirate of the regional lymph nodes, thoracic radiographs and computed tomography scan of the nasal cavity. All dogs were treated with alternating doses of doxorubicin, carboplatin and oral piroxicam. All dogs were monitored for side effects of chemotherapy and evaluated for response to treatment by computed tomography scan of the nasal cavity after the first four treatments. Results Complete remission was achieved in four dogs, partial remission occurred in two dogs and two had stable disease on the basis of computed tomography evaluation. There was resolution of clinical signs after one to two doses of chemotherapy in all dogs. Conclusions This chemotherapy protocol was efficacious and well tolerated in this series of eight cases of canine nasal tumours.

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes

Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R-Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrLS67 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes (R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvA567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R-Avr protein interaction and recapitulate the interaction specificity observed in planta. Biochemical analysis of Escherichia coli-produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R-Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant-pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes.

Improper organization of the actin cytoskeleton affects protein synthesis at initiation

Although the actin cytoskeleton and the translation machinery are considered to be separate cellular complexes, growing evidence supports overlapping regulation of the two systems. Because of its interaction with actin, the eukaryotic translation elongation factor 1A (eEF1A) is proposed to be a regulator or link between these processes. Using a genetic approach with the yeast Saccharomyces cerevisiae, specific regions of eEF1A responsible for actin interactions and bundling were identified. Five new mutations were identified along one face of eEF1A. Dramatic changes in cell growth, cell morphology, and actin cable and patch formation as well as a unique effect on total translation in strains expressing the F308L or S405P eEF1A mutant form were observed. The translation effects do not correlate with reduced translation elongation but instead include an initiation defect. Biochemical analysis of the eEF1A mutant forms demonstrated reduced actin-bundling activity in vitro. Reduced total translation and/or the accumulation of 80S ribosomes in strains with either a mutation or a null allele of genes encoding actin itself or actin-regulating proteins Tpm1p, Mdm20p, and Bnirp/Bni1p was observed. Our data demonstrate that eEF1A, other actin binding proteins, and actin mutants affect translation initiation through the actin cytoskeleton.

The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition

Bacteriophage T7 DNA primase recognizes 5'-GTC-3' in single-stranded DNA. The primase contains a single Cys4 zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys4 motif contacts cytosine of 5'-GTC-3' and may also contribute to thymine recognition. Residues His33 and Asp31 are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5'-GTC-3', by base pairing. Our deduced mechanism for recognition of ssDNA by Cys4 motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys2His2 zinc fingers.

Bacterial associations with salmonid eggs

Rainbow trout eggs Salmo gairdneri, Richardson, were incubated under a range of different environmental conditions. Recovery of bacteria from egg surfaces revealed that increased water temperature, slow water flow rates and high egg density all significantly increased egg surface bacterial populations. Live eggs were mainly colonized by Cytophaga sp., pseudomonas fluorescens and Aeromonas hydrophila. In contrast, dead eggs supported considerable numbers of fluorescent Pseudomonas sp. Analysis of potential nutrient sources for bacteria colonizing live egg surfaces revealed that small amounts of amino acids, phosphate and potassium may be lost by incubating eggs. Subsequently these nutrients were shown to be capable of supporting limited bacterial growth and reproduction. Dead eggs `leaked' increased amounts of the above nutrients which in turn supported higher bacterial numbers. In addition, biochemical analysis of eggs revealed amino acids and fatty acids that might be utilized by bacteria colonizing dead egg surfaces. Assessment of adhesion properties of bacteria frequently recovered from egg surfaces revealed high cell surface hydrophobicity as an important factor in successful egg colonization. Analysis of egg mortalities from groups of rainbow trout and brown trout (S.trutta L.) eggs maintained under two different incubation systems revealed that potentially a close correlation existed between egg surface bacterial numbers and mortalities in the egg during incubation. Innoculation of newly-fertilized eggs with bacteria demonstrated that groups of eggs supporting high numbers of P.fluorescens suffered significantly higher mortalities during the early part of their incubation. Exposure of incubating eggs to oxolinic acid, chlortetracycline and chloramphenicol demonstrated that numbers of bacteria on egg surfaces could be significantly reduced. However, as no corresponding increase in egg hatching success was revealed, the treatment of incubating eggs with antibiotics or antimicrobial compounds can not be recommended. In commercial hatcheries bacteria are only likely to be responsible for egg deaths during incubation when environmental conditions are unfavourable. High water temperatures, slow water flow rates and high egg density all lead to increased bacterial number of egg surfaces, reduced water circulation and low levels of dissolved oxygen. Under such circumstances sufficient amounts of dissolved oxygen may not be available to support developing embryos.

Biomimetic synthetic lubricants (Biosurfactants)

Poly(styrene-co-maleic anhydride) (PSMA) based copolymers are known to undergo conformational transition in response to environmental stimuli. This smart behaviour makes it possible to mimic the behaviour of native apoproteins. The primary aim of this study was to develop a better understanding of the structure-property relationships of various PSMA-based copolymers sought. The work undertaken in this thesis has revealed that the responsive behaviour of PSMA-based copolymers can be tailored by varying the molecular weight, hydrophobic (styrene) and hydrophilic (maleic acid) balance, and more so in the presence of additional hydrophobic, mono-partial ester moieties. Novel hydrophilic and hydrophobic synthetic surfactant protein analogues have successfully been prepared. These novel lipid solubilising agents possess a broad range of HLB (hydrophilic-lipophilic balance) values that have been estimated. NMR spectroscopy was utilised to confirm the structures for PSMA-based copolymers sought and proved useful in furthering understanding of the structure-property relationships of PSMA-based copolymers. The association of PSMA with the polar phospholipid, 2-dilauryl-sn-glycero-3- phosphocholine (DLPC) produces polymer-lipid complexes analogous to lipoprotein assemblies present in the blood plasma. NMR analysis reveals that the PSMA-based copolymers are not perfectly alternating. Regio-irregular structures, atactic and random monomer sequence distribution have been identified for all materials studied. Novel lipid solubilising agents (polyanionic surfactants) have successfully been synthesised from a broad range of PSMA-based copolymers with desired estimated HLB values that interact with polar phospholipids (DLPC/DPPC) uniquely. Very low static and dynamic surface tensions have been observed via the du Noϋy ring method and Langmuir techniques and correlate well with the estimated HLB values. Synthetic protein-lipid analogues have been successfully synthesised, that mimic the unique surface properties of native biological lubricants without the use of solvents. The novel PSMA-DLPC complexes have successfully been combined with hyaluronan (hyaluronic acid, HA). Today, the employment of HA is economically feasible, because it is readily available from bacterial fermentation processes in a thermally stable form - HyaCare®. The work undertaken in this thesis highlights the usage of HA in biolubrication applications and how this can be optimised and thus justified by carefully selecting the biological source, concentration, molecular weight, purity and most importantly by combining it with compatible boundary lubricating agents (polar phospholipids). Experimental evidence supports the belief that the combined HA and PSMA-DLPC complexes provide a balance of rheological, biotribological and surface properties that are composition dependent, and show competitive advantage as novel synthetic biological lubricants (biosurfactants).

Effect of the environment on condition and quality of cultured blue mussels (Mytilus edulis) with reference to culture depth and post-harvest practices

The condition and quality of cultured blue mussels (Mytilus edulis) are affected by various environmental characteristics including temperature, salinity, food concentration, composition and year-to-year variability, waves, tides, and currents. Mussels are a keystone species in the ecosystem, affecting the surrounding environment through filtration, biodeposition and nutrient recycling. This study evaluated the effects of culture depth and post-harvest handling on cultured blue mussels in Newfoundland, Canada. Depth was examined over two years; three shallow water (5 m depth) and three deep water sites (15 m depth) were compared for environmental characteristics, mussel physiological stress response, growth, and biochemical composition. The area examined presented complex hydrodynamic characteristics; deep water sites appeared to be located more often near or within the pycnocline than shallow water sites. Deep water sites presented lower temperatures than shallow sites from spring to fall. Physiological stress response varied seasonally, but was unaffected by culture depth. In Year 1 shallow and deep water mussels presented similar growth, while in Year 2 deep water mussels showed better final condition. Lipid and glycogen showed seasonal variation, but no significant differences between shallow and deep water were noted. Fatty acid profiles showed a higher content of omega-3s PUFA in deep water sites at the end of Year 2. Under extreme weather conditions, deep water appeared to provide a more stable environment for mussel growth than shallow water. Harvested mussels were kept under ambient live-holding conditions for one month during the fall, winter, and spring seasons. They were compared to freshly harvested mussels for condition, biochemical profile and palatability. A progressive loss of dry tissue weight and an increase in water content were shown over the holding period during the fall and spring seasons, when compared to field controls. The biochemical analysis suggested seasonal changes; differences in triacylglycerol content were found in the spring season when compared with controls. The palatability data indicated that the panellists were unable to determine a difference between mussels kept in holding and those freshly harvested from the site. This study presents new knowledge for mussel farming, especially in terms of environmental interactions and deep water culture.

Global distributions of diazotrophs Gamma-A nifH genes abundance - Depth integrated values computed from a collection of source datasets - Contribution to the MAREDAT World Ocean Atlas of Plankton Functional Types

The MAREDAT atlas covers 11 types of plankton, ranging in size from bacteria to jellyfish. Together, these plankton groups determine the health and productivity of the global ocean and play a vital role in the global carbon cycle. Working within a uniform and consistent spatial and depth grid (map) of the global ocean, the researchers compiled thousands and tens of thousands of data points to identify regions of plankton abundance and scarcity as well as areas of data abundance and scarcity. At many of the grid points, the MAREDAT team accomplished the difficult conversion from abundance (numbers of organisms) to biomass (carbon mass of organisms). The MAREDAT atlas provides an unprecedented global data set for ecological and biochemical analysis and modeling as well as a clear mandate for compiling additional existing data and for focusing future data gathering efforts on key groups in key areas of the ocean. The present data set presents depth integrated values of diazotrophs Gamma-A nifH genes abundance, computed from a collection of source data sets.

Global distributions of diazotrophs abundance, biomass and nitrogen fixation rates - Collection of source datasets - Contribution to the MAREDAT World Ocean Atlas of Plankton Functional Types

The MAREDAT atlas covers 11 types of plankton, ranging in size from bacteria to jellyfish. Together, these plankton groups determine the health and productivity of the global ocean and play a vital role in the global carbon cycle. Working within a uniform and consistent spatial and depth grid (map) of the global ocean, the researchers compiled thousands and tens of thousands of data points to identify regions of plankton abundance and scarcity as well as areas of data abundance and scarcity. At many of the grid points, the MAREDAT team accomplished the difficult conversion from abundance (numbers of organisms) to biomass (carbon mass of organisms). The MAREDAT atlas provides an unprecedented global data set for ecological and biochemical analysis and modeling as well as a clear mandate for compiling additional existing data and for focusing future data gathering efforts on key groups in key areas of the ocean. The present collection presents the original data sets used to compile Global distributions of diazotrophs abundance, biomass and nitrogen fixation rates