Steric hindrance controls pyridine nucleotide specificity of a flavin‐dependent NADH:quinone oxidoreductase

The crystal structure of the NADH:quinone oxidoreductase PA1024 has been solved in complex with NAD⁺ to 2.2 Å resolution. The nicotinamide C4 is 3.6 Å from the FMN N5 atom, with a suitable orientation for facile hydride transfer. NAD⁺ binds in a folded conformation at the interface of the TIM‐barrel domain and the extended domain of the enzyme. Comparison of the enzyme‐NAD⁺ structure with that of the ligand‐free enzyme revealed a different conformation of a short loop (75–86) that is part of the NAD⁺‐binding pocket. P78, P82, and P84 provide internal rigidity to the loop, whereas Q80 serves as an active site latch that secures the NAD⁺ within the binding pocket. An interrupted helix consisting of two α‐helices connected by a small three‐residue loop binds the pyrophosphate moiety of NAD⁺. The adenine moiety of NAD⁺ appears to π–π stack with Y261. Steric constraints between the adenosine ribose of NAD⁺, P78, and Q80, control the strict specificity of the enzyme for NADH. Charged residues do not play a role in the specificity of PA1024 for the NADH substrate.     

Effect of curcumin-nanoemulsion associated with photodynamic therapy in breast adenocarcinoma cell line

Curcumin, a natural compound has several antineoplastic activities and is a promising natural photosensitizer used in photodynamic therapy. However, its low solubility in physiological medium limit the clinical use of curcumin. This study aimed to analyze the action of curcumin-nanoemulsion, a new and well-designed Drug Delivery System (DDS+) molecule, used as a photosensitizing agent in photodynamic therapy in an in vitro breast cancer model, MCF-7 cells. The empty nanoemulsion fulfils all necessary requirements to be an excellent DDS. Furthermore, the use of curcumin-nanoemulsion in photodynamic therapy resulted in a high phototoxic effect after activation at 440?nm, decreasing to <10% viable tumor cells after two irradiations and increasing the reactive oxygen species (ROS) production. The use of curcumin-nanoemulsion associated with photodynamic therapy resulted in an increase in the levels of caspase 3/7 activity for the studied MCF-7 cell model, indicating that this therapy triggers a cascade of events that lead to cell death, such as cellular apoptosis. In conclusion, curcumin-nanoemulsion proved to be efficient as a photosensitizing agent, had phototoxic effects, significantly decreased the proliferation of MCF-7 cells and stimulating the ROS production in combination with photodynamic therapy, so, this formulation has a great potential for use in treatment of breast cancer.     

Antimicrobial and antioxidative potential of free and immobilised cellobiose dehydrogenase isolated from wood degrading fungi

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC₅₀ (10.04 ± 0.75 $\text{μg}$/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 $\text{μg}$/ml). The EC₅₀value reached 12.6 ± 1.51 $\text{μg}$/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.     

Acid-catalyzed steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol

Utilization of ethanol produced from biomass has the potential to offset the use of gasoline and reduce CO(2) emissions. This could reduce the effects of global warming, one of which is the current outbreak of epidemic proportions of the mountain pine beetle (MPB) in British Columbia (BC), Canada. The result of this is increasing volumes of dead lodgepole pine with increasingly limited commercial uses. Bioconversion of lodgepole pine to ethanol using SO(2)-catalyzed steam explosion was investigated. The optimum pretreatment condition for this feedstock was determined to be 200 degrees C, 5 min, and 4% SO(2) (w/w). Simultaneous saccharification and fermentation (SSF) of this material provided an overall ethanol yield of 77% of the theoretical yield from raw material based on starting glucan, mannan, and galactan, which corresponds to 244 g ethanol/kg raw material within 30 h. Three conditions representing low (L), medium (M), and high (H) severity were also applied to healthy lodgepole pine. Although the M severity conditions of 200 degrees C, 5 min, and 4% SO(2) were sufficiently robust to pretreat healthy wood, the substrate produced from beetle-killed (BK) wood provided consistently higher ethanol yields after SSF than the other substrates tested. BK lodgepole pine appears to be an excellent candidate for efficient and productive bioconversion to ethanol.     

A comparative study of different glycosylation methods for the synthesis of D-mannopyranosides of Nalpha-fluorenylmethoxycarbonyl-trans-4-hydroxy-L-proline allyl ester

The synthesis of Nalpha-fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-O-acetyl)-alpha-d-mannopyranosyl]-l-proline allyl ester and Nalpha-fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-O-benzoyl)-alpha-d-mannopyranosyl]-l-proline allyl ester is described. Glycosylation using K?nigs-Knorr conditions with a benzoyl protected glycosyl donor provided the optimum method. Removal of the allyl ester gave two mannosylated building blocks suitable for solid phase glycopeptide synthesis.     

Long-term feeding a high-fat diet causes histological and parasitological effects on murine schistosomiasis mansoni outcome

This study investigated whether long-term feeding a high-fat diet (HFC) has an effect on schistosomiasis mansoni outcome compared to standard chow diet (SC). Swiss Webster female mice (3 wk old) fed each diet over 5 months, and then were infected with 50 Schistosoma mansoni cercariae. Their nutritional status was assessed by monitoring growth rates twice a week and measuring serum levels of lipoproteins. Mice were euthanised 63 days after infection. Parasitological and liver histological analyses were performed. The levels of TC, HDL-C and LDL-C, fecal and tissue schistosome eggs were statistically different (p<0.05) between groups. Livers from HFC mice showed exudative, exudative/exudative-productive, exudative-productive and productive granulomas, some degree of hepatic steatosis and focal necrosis. Mice fed normal-chow did not present productive granulomas and hepatic steatosis. The morphometric evaluation of hepatic granulomas did not reach statistical significance (p>0.05) between diets assayed. The high-fat diet for long-term produces effects on schistosomiasis mansoni outcome.     

Serotonin increases the phase shift of the circadian locomotor activity rhythm in mice after dark pulses in constant light conditions

Investigations on the effects of the 5-HT agonists and antagonists on the phase of the circadian locomotor activity rhythm of animals kept in constant light conditions (LL) are rare. Therefore the influence of R-(+)-OH-DPAT (5-HT1A receptors agonist) and metergoline (5-HT1/2/7 receptors antagonist) on the phase shift of the locomotor-activity rhythm alone and when combined with dark pulses in mice kept in LL are examined. The results indicate that 8-OH-DPAT administered independently at 12.00CT (Circadian Time) shifted the phase of the circadian rhythm and reinforced the effect of dark pulses on this parameter. 12.00CT was defined arbitrarily as the onset of locomotor activity in constant conditions. Metergoline diminished the phase shifts after dark pulses compared to 8-OH-DPAT. The influence of the serotonin agonist showed that serotonin can reinforce the phase shifting effect of the locomotor activity rhythm after dark pulses in LL condition.     

SCLIP, a microtubule-destabilizing factor, interacts with RasGRF1 and inhibits its ability to promote Rac activation and neurite outgrowth

RasGRF1 is a neuron-specific guanine nucleotide exchange factor for the small GTPases Ras and Rac. It is implicated in the regulation of memory formation and in the development of tolerance to drug abuse, although the mechanisms have been elucidated only in part. Here we report the isolation, by the yeast two-hybrid screen, of the microtubule-destabilizing factor SCLIP (SCG10-like protein) as a novel RasGRF1-interacting protein. This interaction requires the region spanning the Dbl-homology domain of RasGRF1, endowed with catalytic activity on Rac. In search for a possible function we found by biochemical means that SCLIP influences the signaling properties of RasGRF1, greatly reducing its ability to activate the Rac/p38 MAPK pathway, while the Ras/Erk one remains unaffected. Moreover, a potential role is suggested by transfection studies in neuronal PC12 cells in which RasGRF1 induces neurite outgrowth, and coexpression of SCLIP counteracts this effect, causing a dramatic decrease in the percentage of cells bearing neurites, which also appear significantly shortened. This study unveils a physical and functional interaction between RasGRF1 and SCLIP. We suggest that this novel interplay may have possible implications in mechanisms that regulate neuronal morphology and structural plasticity.     

Molecular PET imaging of HSV1-tk reporter gene expression using [18F]FEAU

Non-invasive imaging of transgene expression requires the appropriate combination of a reporter gene and a reporter probe. [18F]FEAU positron emission tomography (PET) is used for the assessment of herpes simplex virus type-1 thymidine kinase gene expression. Hybrid AAV phage (termed AAVP) can be adapted to transduce mammalian cells by targeting to a specific receptor. We evaluated a targeted AAVP vector using [18F]FEAU PET. This protocol describes [18F]FEAU production and dosing, micro-PET imaging and image analysis. 2-Deoxy-2-trifluoromethanesulfonyl-1,3,5-tri-O-benzoyl-alpha-D-ribofuranose is radio-fluorinated, converted into its 1-bromo derivative and coupled with protected 5-ethyl uracil. The coupled product is hydrolyzed and purified using HPLC. Tumor-bearing animals targeted with either retroviral or AAVP vectors are anesthetized and injected with [18F]FEAU (0.1 mCi per mouse); this is followed 2 h after injection by imaging on a micro-PET. Production of [18F]FEAU requires approximately 3.5 h from the end of bombardment. PET imaging studies require 2-3 h (depending on the number of animals) after synthesis of [18F]FEAU.