Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

Parkinson’s disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by α-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z′ factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD.     

Relationship Between Molecular Structure and Thermo-mechanical Properties of Candelilla Wax and Amides Derived from (<Emphasis Type="Italic">R</Emphasis>)-12-Hydroxystearic Acid as Gelators of Safflower Oil

In this research, we studied the relationship between the molecular structure of (R)-12-hydroxyoctadecanamide, (R)-N-propyl-12-hydroxyoctadecanamide, and (R)-N-octadecyl-12-hydroxyoctadecanamide and the thermo-mechanical properties of their 2% (wt/wt) organogels developed using safflower oil high in oleic acid (HOSFO) as the liquid phase. Candelilla wax (CW), a well-known edible gelling additive whose main component is hentriacontane, also was studied for comparative purposes. The results obtained show that the attractive interactions (i.e., hydrogen bonding and dipolar interactions) between amide groups and between hydroxyl groups present in the amides resulted in organogels with higher melting temperature, heat of melting, and crystallization parameters than those found in the CW organogel. The rheological parameters associated to the strength of the amide or CW-based gels developed in HOSFO (i.e., yield stress and elastic modulus) seem to be associated with the nature of amide groups (i.e., primary or secondary amide) and the increase in the length of the self-assembly molecular unit (i.e., L value determined by X-ray diffraction) and therefore to the extent of London dispersion forces along the hydrocarbon chain. The creep and recovery measurements allowed an evaluation among the internal structures of the different organogels and demonstrated that independent of the hydrogen bonding and dipolar interaction provided by the amide and the hydroxyl groups, the increase in the hydrocarbon chain length results in higher organogel resistance to deformation and higher instant recovery capacity. However, the stabilization of the self-assembly unit through polar groups (i.e., –CONH₂ in HOA) reduces organogel elasticity but provides a higher extended recovery capacity. The results reported in this investigation showed some relationships between gelator structure and the thermo-mechanical properties of low-molecular-mass organic gelator amides. Our long-term objective is to understand the organogelation process to eventually develop trans-free vegetable oil-based food products with novel textures for the consumers.     

Potential Interaction of Plasmodium falciparum Hsp60 and Calpain

After invasion of red blood cells, malaria matures within the cell by degrading hemoglobin avidly. For enormous protein breakdown in trophozoite stage, many efficient and ordered proteolysis networks have been postulated and exploited. In this study, a potential interaction of a 60-kDa Plasmodium falciparum (Pf)-heat shock protein (Hsp60) and Pf-calpain, a cysteine protease, was explored. Pf-infected RBC was isolated and the endogenous Pf-Hsp60 and Pf-calpain were determined by western blot analysis and similar antigenicity of GroEL and Pf-Hsp60 was determined with anti-Pf-Hsp60. Potential interaction of Pf-calpain and Pf-Hsp60 was determined by immunoprecipitation and immunofluorescence assay. Mizoribine, a well-known inhibitor of Hsp60, attenuated both Pf-calpain enzyme activity as well as P. falciparum growth. The presented data suggest that the Pf-Hsp60 may function on Pf-calpain in a part of networks during malaria growth.     

Animal Welfare in Relation to Standards in Organic Farming

The new EU-regulations on organic farming (1804/1999) are also influencing the animal welfare. A lot of positive regulations is to find, but also regulations that seen to mind more about the general public and customer and their view on organic farming, than the health and welfare of the animals.The paper specially focus on the impact of the regulations and the recommendations that phytotherapeutic essences and homeopathic products take precedence over the so called chemically-synthesised allopatic veterinary medical products, and that the use of the same is prohibited for preventive treatments. Key questions here are the lack of scientific evidence concerning homeopathy in animals, and that Swedish veterinarians are not allowed to work with homeopathy.Differences in interpretation of the regulations between animal owners and veterinarians will also be discussed. What is a disease that needs treatment? Who is to decide about the treatment? Parasitic infections are discussed as an illustrative example.Other consequences of the regulations concerning the animal welfare are problems in certain geographical zones, for instance subarctic areas where necessary crops are impossible to grow.Animal transports and splitting mother-offspring are briefly discussed as future problems to be handled in the regulations, and the paper ends by presenting the need of regulated herd health control programs in organic husbandry, which can detect and focus on welfare and production problems. The organic movement is not static, and must not be so.     

Reduced Medication in Organic Farming with Emphasis on Organic Dairy Production

Medication is an important focus area in organic animal husbandry. The combination of goals relating to improved animal welfare and reduced use of chemicals in general creates a common wish to reduce medication. Based on data from current Danish research projects in organic dairy farming, one specific organic medication pattern or policy cannot be described. The disease treatment pattern is influenced by many factors, e.g. the interaction with colleagues, veterinarians and agricultural advisors. No significant difference could be found with regard to incidence of mastitis treatments or somatic cell counts in 27 organic and 57 conventional herds. A marked tendency to shorter treatment periods in relation to mastitis treatments was described for organic farms in comparison with conventional farms (1.9 days versus 3.2 days (5 organic and 7 conventional herds)). In a study of development of health advisory service in organic herds, the dialogue between farmer, veterinarian and agricultural cattle advisor changed the treatment pattern markedly during a period of 6 months. Among important future challenges for veterinarians in organic farming is pointed at the constructive, open, and critical interaction with the single organic farmer as well as the organic animal husbandry system in general.     

Complex phylogenetic distribution of a non-canonical genetic code in green algae

Background  

A non-canonical nuclear genetic code, in which TAG and TAA have been reassigned from stop codons to glutamine, has evolved independently in several eukaryotic lineages, including the ulvophycean green algal orders Dasycladales and Cladophorales. To study the phylogenetic distribution of the standard and non-canonical genetic codes, we generated sequence data of a representative set of ulvophycean green algae and used a robust green algal phylogeny to evaluate different evolutionary scenarios that may account for the origin of the non-canonical code.     

Cyclic AMP increases the Adhesion of Fibroblasts to Substratum

THE interaction between the cell surface and the substratum is very important in determining several characteristics of cells growing in tissue culture. Transformed cells are less adherent to the substratum than untransformed cells¹ and this reduced interaction with the substratum may be responsible for abnormal properties such as the loss of contact or density dependent inhibition of growth² and the ability to form colonies in agar and to grow in suspension culture.     

Retinol (Vitamin A) Increases α-Synuclein, β-Amyloid Peptide,Tau Phosphorylation and RAGE Content in Human SH-SY5Y Neuronal Cell Line

Retinoids (vitamin A and derivatives) are recognized as essential factors for central nervous system (CNS) development. Retinol (vitamin A) also was postulated to be a major antioxidant component of diet as it modulates reactive species (RS) production and oxidative stress in biological systems. Oxidative stress plays a major role either in pathogenesis or development of neurodegenerative diseases, or even in both. Here we investigate the role of retinol supplementation to human neuron-derived SH-SY5Y cells over RS production and biochemical markers associated to neurodegenerative diseases expressed at neuronal level in Parkinson’s disease and Alzheimer’s disease: α-synuclein, β-amyloid peptide, tau phosphorylation and RAGE. Retinol treatment (24 h) impaired cell viability and increased intracellular RS production at the highest concentrations (7 up to 20 µM). Antioxidant co-treatment (Trolox 100 µM) rescued cell viability and inhibited RS production. Furthermore, retinol (10 µM) increased the levels of α-synuclein, tau phosphorylation at Ser396, β-amyloid peptide and RAGE. Co-treatment with antioxidant Trolox inhibited the increased in RAGE, but not the effect of retinol on α-synuclein, tau phosphorylation and β-amyloid peptide accumulation. These data indicate that increased availability of retinol to neurons at levels above the cellular physiological concentrations may induce deleterious effects through diverse mechanisms, which include oxidative stress but also include RS-independent modulation of proteins associated to progression of neuronal cell death during the course of neurodegenerative diseases.     

Focal adhesion kinase maintains,but not increases the adhesion of dental pulp cells

Focal adhesion kinase (FAK) functions as a key enzyme in the integrin-mediated adhesion-signalling pathway. Here, we aimed to investigate the effects of FAK on adhesion of human dental pulp (HDP) cells. We transfected lentiviral vectors to silence or overexpress FAK in HDP cells ex vivo. Early cell adhesion, cell survival and focal contacts (FCs)-related proteins (FAK and paxillin) were examined. By using immunofluorescence, the formation of FCs and cytoskeleton was detected, respectively. We found that both adhesion and survival of HDP cells were suppressed by FAK inhibition. However, FAK overexpression slightly inhibited cell adhesion and exhibited no change in cell survival compared with the control. A thick rim of cytoskeleton accumulated and smaller dot-shaped FCs appeared in FAK knockdown cells. Phosphorylation of paxillin (p-paxillin) was inhibited in FAK knockdown cells, verifying that the adhesion was inhibited. Less cytoskeleton and elongated FCs were observed in FAK-overexpressed cells. However, p-paxillin had no significant difference compared with the control. In conclusion, the data suggest that FAK maintains cell adhesion, survival and cytoskeleton formation, but excessive FAK has no positive effects on these aspects.