Enzymatic pre‐treatment of microalgae cells for enhanced extraction of proteins

Crude proteins and pigments were extracted from different microalgae strains, both marine and freshwater. The effectiveness of enzymatic pre‐treatment prior to protein extraction was evaluated and compared to conventional techniques, including ultrasonication and high‐pressure water extraction. Enzymatic pre‐treatment was chosen as it could be carried out at mild shear conditions and does not subject the proteins to high temperatures, as with the ultrasonication approach. Using enzymatic pre‐treatment, the extracted proteins yields of all tested microalgae strains were approximately 0.7 mg per mg of dry cell weight. These values were comparable to those achieved using a commercial lytic kit. Ultrasonication was not very effective for proteins extraction from Chlorella sp., and the extracted proteins yields did not exceed 0.4 mg per mg of dry cell weight. For other strains, similar yields were achieved by both treatment methods. The time‐course effect of enzymatic incubation on the proteins extraction efficiency was more evident using laccase compared to lysozyme, which suggested that the former enzyme has a slower rate of cell disruption. The crude extracted proteins were fractionated using an ion exchange resin and were analyzed by the electrophoresis technique. They were further tested for their antioxidant activity, the highest of which was about 60% from Nannochloropsis sp. The total phenolic contents in the selected strains were also determined, with Chlorella sp. showing the highest content reaching 17 mg/g. Lysozyme was also found to enhance the extraction of pigments, with Chlorella sp. showing the highest pigments contents of 16.02, 4.59 and 5.22 mg/g of chlorophyll a, chlorophyll b and total carotenoids, respectively.     

Reaction of haem containing proteins and enzymes with hydroperoxides: the radical view

The reaction between hydroperoxides and the haem group of proteins and enzymes is important for the function of many enzymes but has also been implicated in a number of pathological conditions where oxygen binding proteins interact with hydrogen peroxide or other peroxides. The haem group in the oxidized Fe3+ (ferric) state reacts with hydroperoxides with a formation of the Fe4+=O (oxoferryl) haem state and a free radical primarily located on the pi-system of the haem. The radical is then transferred to an amino acid residue of the protein and undergoes further transfer and transformation processes. The free radicals formed in this reaction are reviewed for a number of proteins and enzymes. Their previously published EPR spectra are analysed in a comparative way. The radicals directly detected in most systems are tyrosyl radicals and the peroxyl radicals formed on tryptophan and possibly cysteine. The locations of the radicals in the proteins have been reported as follows: Tyr133 in soybean leghaemoglobin; alphaTyr42, alphaTrp14, betaTrp15, betaCys93, (alphaTyr24-alphaHis20), all in the alpha- and beta-subunits of human haemoglobin; Tyr103, Tyr151 and Trp14 in sperm whale myoglobin; Tyr103, Tyr146 and Trp14 in horse myoglobin; Trp14, Tyr103 and Cys110 in human Mb. The sequence of events leading to radical formation, transformation and transfer, both intra- and intermolecularly, is considered. The free radicals induced by peroxides in the enzymes are reviewed. Those include: lignin peroxidase, cytochrome c peroxidase, cytochrome c oxidase, turnip isoperoxidase 7, bovine catalase, two isoforms of prostaglandin H synthase, Mycobacterium tuberculosis and Synechocystis PCC6803 catalase-peroxidases.     

Reaction of haem containing proteins and enzymes with hydroperoxides: The radical view

The reaction between hydroperoxides and the haem group of proteins and enzymes is important for the function of many enzymes but has also been implicated in a number of pathological conditions where oxygen binding proteins interact with hydrogen peroxide or other peroxides. The haem group in the oxidized Fe³⁺ (ferric) state reacts with hydroperoxides with a formation of the Fe⁴⁺=O (oxoferryl) haem state and a free radical primarily located on the π-system of the haem. The radical is then transferred to an amino acid residue of the protein and undergoes further transfer and transformation processes. The free radicals formed in this reaction are reviewed for a number of proteins and enzymes. Their previously published EPR spectra are analysed in a comparative way. The radicals directly detected in most systems are tyrosyl radicals and the peroxyl radicals formed on tryptophan and possibly cysteine. The locations of the radicals in the proteins have been reported as follows: Tyr133 in soybean leghaemoglobin; αTyr42, αTrp14, βTrp15, βCys93, (αTyr24−αHis20), all in the α- and β-subunits of human haemoglobin; Tyr103, Tyr151 and Trp14 in sperm whale myoglobin; Tyr103, Tyr146 and Trp14 in horse myoglobin; Trp14, Tyr103 and Cys110 in human Mb. The sequence of events leading to radical formation, transformation and transfer, both intra- and intermolecularly, is considered. The free radicals induced by peroxides in the enzymes are reviewed. Those include: lignin peroxidase, cytochrome c peroxidase, cytochrome c oxidase, turnip isoperoxidase 7, bovine catalase, two isoforms of prostaglandin H synthase, Mycobacterium tuberculosis and Synechocystis PCC6803 catalase-peroxidases.     

NQO1-dependent redox cycling of idebenone: effects on cellular redox potential and energy levels

Short-chain quinones are described as potent antioxidants and in the case of idebenone have already been under clinical investigation for the treatment of neuromuscular disorders. Due to their analogy to coenzyme Q10 (CoQ10), a long-chain quinone, they are widely regarded as a substitute for CoQ10. However, apart from their antioxidant function, this provides no clear rationale for their use in disorders with normal CoQ10 levels. Using recombinant NAD(P)H:quinone oxidoreductase (NQO) enzymes, we observed that contrary to CoQ10 short-chain quinones such as idebenone are good substrates for both NQO1 and NQO2. Furthermore, the reduction of short-chain quinones by NQOs enabled an antimycin A-sensitive transfer of electrons from cytosolic NAD(P)H to the mitochondrial respiratory chain in both human hepatoma cells (HepG2) and freshly isolated mouse hepatocytes. Consistent with the substrate selectivity of NQOs, both idebenone and CoQ1, but not CoQ10, partially restored cellular ATP levels under conditions of impaired complex I function. The observed cytosolic-mitochondrial shuttling of idebenone and CoQ1 was also associated with reduced lactate production by cybrid cells from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) patients. Thus, the observed activities separate the effectiveness of short-chain quinones from the related long-chain CoQ10 and provide the rationale for the use of short-chain quinones such as idebenone for the treatment of mitochondrial disorders.     

Murine monoclonal antibody against aldosterone: production, characterization and use for enzymoimmunoassay

Hybridomas secreting monoclonal antibodies to aldosterone were obtained by fusion of myeloma cells and spleen cells from Balb/c mice immunized with aldosterone-3-carboxylmethyloxime-bovine serum albumin. A monoclonal antibody was purified from ascites fluid and characterized. An affinity constant of 1.61 x 10(9) M-1 has been measured and no cross-reactivity with tetrahydroaldosterone (THA), cortisol, cortisone, corticosterone, deoxycorticosterone (DOC), dehydroepiandrosterone (DHA), progesterone and estrone, could be detected. A peroxidase conjugated-antibody (1.5 mole of enzyme per mole of antibody) was obtained and used for microwell enzyme immunoassay and Immun-Blot assay. The high affinity and specificity of this antibody should make the direct determination of aldosterone in biological fluids possible at concentrations as low as 5 x 10(-10) M.     

Inhibition of lymphocyte transformation: effect of soy bean trypsin inhibitor and synthetic anti-proteases.

Soybean trypsin inhibitor (SBI) was found to inhibit transformation of human lymphocytes induced by mitogens (leucoagglutinin, concanavalin A, NaIO4) or in mixed lymphocyte reaction (MLR). SBI covalently cross-linked to Sepharose beads inhibited the MLR and mitogen stimulation virtually completely. We have confirmed the work of others which showed that the synthetic anti-proteases epilson-aminocaproic acid and tosyl-L-lysyl-chloromethane (TLCK) also inhibited mitogen-induced blastogenesis and we have shown that phenylmethylsulfonylfluride was effective also; all of these agents were found to inhibit the MLR as well. SBI and TLCK were most inhibitory when added along with mitogen or when mixing allogeneic cells in a MLR; significant decrease in inhibition was noted when TLCK was added 1 h after mitogen. These data support the hypothesis that protease action at a cell surface is an essential early event common to all types of lymphocyte transformation.     

Vectors for co-expression of an unrestricted number of proteins

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells.     

Bioluminescent signals spatially amplified by wavelength-specific diffusion through the shell of a marine snail

Some living organisms produce visible light (bioluminescence) for intra- or interspecific visual communication. Here, we describe a remarkable bioluminescent adaptation in the marine snail Hinea brasiliana. This species produces a luminous display in response to mechanical stimulation caused by encounters with other motile organisms. The light is produced from discrete areas on the snail's body beneath the snail's shell, and must thus overcome this structural barrier to be viewed by an external receiver. The diffusion and transmission efficiency of the shell is greater than a commercial diffuser reference material. Most strikingly, the shell, although opaque and pigmented, selectively diffuses the blue-green wavelength of the species bioluminescence. This diffusion generates a luminous display that is enlarged relative to the original light source. This unusual shell thus allows spatially amplified outward transmission of light communication signals from the snail, while allowing the animal to remain safely inside its hard protective shell.