Reduced human lymphocyte blastogenesis and enhancement of adenosine triphosphate (ATP) by L-carnitine

The spice principles curcumin (from turmeric) and eugenol (from cloves) are good inhibitors of lipid peroxidation. Lipid peroxidation is known to be initiated by reactive oxygen species. The effect of curcumin and eugenol on the generation of reactive oxygen species in model systems were investigated. Both curcumin and eugenol inhibited superoxide anion generation in xanthine-xanthine oxidase system to an extent of 40% and 50% at concentrations of 75 M and 250 M respectively. Curcumin and eugenol also inhibited the generation of hydroxyl radicals (^.OH) to an extent of 76% and 70% as measured by deoxyribose degradation. The^.OH-radical formation measured by the hydroxylation of salicylate to 2,3-dihydroxy benzoate was inhibited to an extent of 66% and 46%, respectively, by curcumin and eugenol at 50 M and 250 M. These spice principles also prevented the oxidation of Fe²⁺ in Fentons reaction which generates^.OH radicals.     

Relevance of plant lectins in human cell biology and immunology.

Protein-carbohydrate interactions are used for intercellular communication. Mammalian cells are known to bear a variety of glycoconjugates. Lectins, first discovered in plants, are proteins which can specifically bind carbohydrates. Given the high affinity of plant lectins for carbohydrates, they have always been important as molecular tools in the identification, purification and stimulation of specific glycoproteins on human cells. Lectins have provided important clues to the repertoire of carbohydrate structures in animal cells. The discovery of plant lectins gave a great impulse to modern glycobiology. They represent important biochemical reagents for numerous applications in the biomedical field and in research. Sequence determinations and structural characterization helped to understand the mechanism of action in many biologic systems. Plant lectins have been fundamental in human immunological studies because some of them are mitogenic/activating to lymphocytes. Understanding the molecular basis of lectin-carbohydrate interactions and of the intracellular signalling evoked holds promise for the design of novel drugs for the treatment of infectious, inflammatory and malignant diseases. It may also be of help for the structural and functional investigation of glycoconjugates and their changes during physiological and pathological processes.     

Use of electrospray tandem mass spectrometry for identification of microcystins during a cyanobacterial bloom event

Drastic environmental conditions such as elevated temperature, abrupt pH variation, low turbulence, and high nutrient inputs can enhance the development of toxic cyanobacterial blooms in lakes and reservoirs. This study describes the occurrence of four microcystin variants (MC) in a bloom in the eutrophic reservoir Billings, in S?o Paulo City. The bloom sample was collected in October 2003, and Microcystis were the main genus found. The MC were separated and purified by reverse phase high performance liquid chromatography (RP-HPLC). Their structures were elucidated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and four MC variants were determined: MC-RR, MC-LR, MC-YR, and MC-hRhR. MC-hRhR is described for the first time as a new variant of MC with two homoarginines at positions 2 and 4 in its structure. ESI-MS/MS analysis thus provides a powerful and convenient tool for the determination of variants of MC. These results represent an important contribution to the knowledge of the biochemistry of toxic cyanobacteria and their toxins, specifically in S?o Paulo State.     

Direct Fabrication Route to Plastic-Supported Gold Nanoparticles for Flexible NIR-SERS

Stable gold nanoparticles with surface plasmon resonance tunable from visible (Vis) to near-infrared (NIR) are deposited via a direct sputtering methodology on large area polyethylene terephthalate (PET) to be used as effective, flexible NIR surface-enhanced Raman scattering (SERS) substrates. An O₂ plasma treatment of PET is used to tailor growth dynamics, geometry, and plasmonic properties of nanoparticles. The O₂ plasma treatment of PET results also in effective improvement of nanoparticle anchoring on the plastic substrate, providing more stable, flexible SERS systems. The functionality of fabricated SERS substrates has been tested using benzylthiol, and SERS enhancement factors in the range 10⁴ have been achieved, which are comparable with those reported in literature for gold nanostructures fabricated on silicon substrate. These results attest the great potentiality of this methodology for the production of cost-effective flexible and reusable large-scale SERS substrates.     

Circadian Protection against Reactive Oxygen Species Involves Changes in Daily Levels of the Manganese- and Iron-Containing Superoxide Dismutase Isoforms in Lingulodinium polyedrum

A circadian rhythm in the total activity of superoxide dismutase (SOD; EC 1.15.1.1) from the unicellular alga Lingulodinium polyedrum is shown to be attributable to the mitochondrial MnSOD and chloroplastic FeSOD isoforms. Activity gels and labelling with polyclonal antibodies against pure CuZnSOD, MnSOD and FeSOD revealed a distinct circadian pattern in the abundance of the latter two isoforms, with peak values in early photophase 5 times greater than at the dark phase. However, no such changes were detected for the CuZnSOD isoform, which remained at constant levels over the 24-h light/dark cycle. These SOD isoforms might provide protection against damage from photochemically generated oxygen radicals, thus preventing subcellular oxidative stress.     

Oscillation of Reactive Oxygen Species Released by Activated Neutrophils

Phagocytic leukocytes, such as neutrophils and macrophages release reactive oxygen species (ROS) to the surrounding medium upon appropriate stimulation as part of their cytocidal activity. The release of ROS by inflammatory neutrophils, obtained by peritoneal injection of 12% caseinate-PBS was measured by the reduction of ferricytochrome c and luminol chemiluminescence (LCL). Neutrophils were harvested every 4 hours with cold PBS and stimulated with phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). On a regimen providing light between 6:00 to 18:00, PMA-stimulated neutrophils (1.0 x 10 7 neutrophil/ml) were found to release twice as much superoxide anion at night as they did during the day (clock time; 2:00 = 1.43 nmol/min vs. clock time 14:00 = 0.65 nmol/min). Neither FMLP- nor OZ-stimulated neutrophils displayed similar fluctuations. Thus, the qualitative and quantitative aspects of ROS generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the effect of neutrophils on microorganisms and the surrounding tissues may differ with the nature of the stimulus and the time the stimulus is given.     

Contributo Alla Flora Dell'alto Adige

In order to obtain a high ethanol yield from the Jerusalem artichoke raw extract and reduce the fermentation cost, we have engineered a new recombinant Saccharomyces cerevisiae strain that could produce ex-inulinase. The response surface methodology based on Plackett–Burman and Box–Behnken design was used to optimize the medium for the ethanol production from the Jerusalem artichoke raw extracts by the recombinant strain. In the first optimization step, Plackett–Burman design was employed to select significant factors, including concentrations of yeast extract, inoculum, and MgSO₄·7H₂O. In the second step, the steepest ascent experiment was carried out to determine the center point with the three significant factors; the selected combinations were further optimized using the Box–Behnken design. The maximum ethanol production rate was predicted at 91.1 g/l, which was based on a medium consisting of yeast extract 9.24 g/l, inoculum 39.8 ml/l, and MgSO₄·7H₂O 0.45 g/l. In the validating experiment, the ethanol fermentation rate reached 102.1 g/l, closely matching the predicted rate.     

Low cryoprotectant concentrations and fast cooling for nematode cryostorage

Cryopreservation protocols based on slow freezing or vitrification often result in cell injury due to ice formation, cell dehydration and/or toxic concentrations of cryoprotectant (CPA).In this study, we present a cryopreservation technique based on low, non-toxic concentrations of cryoprotectants (≈2–4 M) combined with a rapid cooling rate in the liquid nitrogen phase (−196 °C). Protocols for successfully cryopreserving the plant parasitic nematodes Globodera tabacum tabacum, Heterodera schachtii and Meloidogyne incognita were developed, as demonstrated by the high survival rates and reproducibility of cyst and root-knot nematode species post-cryostorage. This approach for effective cryopreservation of viable plant-parasitic nematodes was developed by inducing an “apparent vitrification” by rapid cooling of the microscopic samples in less than 2 M of cryoprotectant. The extremely thin structure (15–20 μm width, 350–400 μm length) of these nematodes, in combination with a direct and rapid exposure to LN₂, likely prevents the formation of damaging ice crystals. Moreover, this procedure results in viability of both short- and long-cryostorage samples. These techniques could potentially be used for the near-indefinite preservation of thousands of different nematode species. A cryo-nematode collection produced in our lab is available and presented here.     

Long-term intermittent feeding, but not caloric restriction, leads to redox imbalance, insulin receptor nitration, and glucose intolerance

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance.