Application of the SMALP technology to the isolation of GPCRs from low-yielding cell lines

The ability of styrene–maleic acid (SMAc) co-polymers to spontaneously insert into biological membranes can be exploited to extract G protein-coupled receptors (GPCRs) embedded in styrene–maleic acid lipid particles (SMALPs), preserving the native environment around the protein and thus enhancing the feasibility of functional studies. So far, the SMALP technology has been primarily employed on non-mammalian cells and protocols are not optimized for adherent human cell lines, which cannot be harvested in large amounts. In this work, a fine investigation of key parameters affecting the formation of SMALPs was undertaken with the purpose of maximizing the yield of extraction of a recombinant form of human β2-adrenergic receptor (rhβ2AR) from HEK293T cells. The study highlighted an important influence of ionic strength on the membrane solubilization efficiency and GPCR purification yield of SMAc co-polymers: by lowering the salt concentration of all buffers used in previously published SMALP protocols, the water solubility and extraction efficiency of the selected SMAc co-polymer (commercially supplied as a potassium salt) were enhanced. In-line combination of size-exclusion chromatography (SEC) with immobilized metal affinity chromatography (IMAC) allowed further improvement of the final rhβ2AR yield by reducing the loss of SMALP-embedded GPCRs during the fractionation and purification of SMALPs. The overall findings of this study show that the available SMALP protocols can be significantly optimized in several aspects in order to increase the efficiency of GPCR solubilization and isolation from low-yielding expression systems

Genes Associated with 2-Methylisoborneol Biosynthesis in Cyanobacteria: Isolation, Characterization, and Expression in Response to Light

The volatile microbial metabolite 2-methylisoborneol (2-MIB) is a root cause of taste and odor issues in freshwater. Although current evidence suggests that 2-MIB is not toxic, this compound degrades water quality and presents problems for water treatment. To address these issues, cyanobacteria and actinomycetes, the major producers of 2-MIB, have been investigated extensively. In this study, two 2-MIB producing strains, coded as Pseudanabaena sp. and Planktothricoids raciborskii, were used in order to elucidate the genetic background, light regulation, and biochemical mechanisms of 2-MIB biosynthesis in cyanobacteria. Genome walking and PCR methods revealed that two adjacent genes, SAM-dependent methyltransferanse gene and monoterpene cyclase gene, are responsible for GPP methylation and subsequent cyclization to 2-MIB in cyanobacteria. These two genes are located in between two homologous cyclic nucleotide-binding protein genes that may be members of the Crp-Fnr regulator family. Together, this sequence of genes forms a putative operon. The synthesis of 2-MIB is similar in cyanobacteria and actinomycetes. Comparison of the gene arrangement and functional sites between cyanobacteria and other organisms revealed that gene recombination and gene transfer probably occurred during the evolution of 2-MIB-associated genes. All the microorganisms examined have a common origin of 2-MIB biosynthesis capacity, but cyanobacteria represent a unique evolutionary lineage. Gene expression analysis suggested that light is a crucial, but not the only, active regulatory factor for the transcription of 2-MIB synthesis genes. This light-regulated process is immediate and transient. This study is the first to identify the genetic background and evolution of 2-MIB biosynthesis in cyanobacteria, thus enhancing current knowledge on 2-MIB contamination of freshwater

The mechanism and kinetics of ozone formation in a pulse radiolysis study of the Ar-O2 system

The mechanism and kinetics of O3 formation after an electron pulse have been studied in the Ar-O2 systems by time resolved optical measurements at gamma = 260 nm. The second order rate constant of energy transfer from excited Ar(4s,4p) states to O2 molecules: (1) Ar*,Ar** + O2 → O2* + Ar, was found to be (8.9 ± 2.1) x 10-10 cm3 s-1. It was found also the evidence of the third order process contribution to the energy transfer: (2) Ar*,Ar** + Ar + O2 → products, with the rate constant in the range (1.5-3.7) x 10-29 cm6 s-1. The rate constant of the deactivation of excited ozone molecules by O2 was found to be (5.1 ± 0.6) ´ 10-15 cm3 s-1

Problems associated with the presence of cyanobacteria in recreational and drinking waters

Eutrophication of waters can lead to the development of blooms of cyanobacteria (blue-green algae) and consequent health and environmental problems. The presence of these organisms in recreational and drinking waters is generally undesirable or even hazardous, although nitrogen fixing blue-green algae can be beneficially used as biofertilisers for plantation crops. This paper reviews the characteristics of cyanobacteria and particularly their toxins. The mechanisms of toxic algal blooms are discussed, as are the factors influencing toxin production. The nuisance and health hazards associated with freshwater bluegreen algae are discussed and the options for public health control are evaluated. The problems associated with statutory control of toxic algae problems is also considered