A set of epitope-tagging integration vectors for functional analysis in Saccharomyces cerevisiae

Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.     

Radiolytic transformation of rotenone with potential anti-adipogenic activity

Radiolytic transformation of the isoflavonoid rotenone (1) with γ-irradiation afforded two new degraded products, rotenoisins A (2) and (3). The structures of the two new rotenone derivatives were elucidated on the basis of spectroscopic methods. The new products 2 and 3 exhibited significantly enhanced inhibitory activities against pancreatic lipase and adipocyte differentiation in 3T3-L1 cells when compared to parent rotenone.     

Pharmacophore-based strategy for the development of general and specific scFv biosensors for abused antibiotics

We developed fluorescent biosensor systems that are either general or selective to fluoroquinolone antibiotics by using a single-chain variable-fragment (scFv) as a recognition element. The selectivity of these biosensors to fluoroquinolone antibiotics was rationally tuned through the structural modification on the pharmacophore of fluoroquinolone antibiotics and the subsequent selection of scFv receptor modules against these antibiotics-based antigens using phage display. The resulting A2 and F9 scFv's bound to their representative antigen with a moderate affinity (K(D) in micromolar range as determined by surface plasmon resonance). A2 is a specific binder for enrofloxacin and did not cross-react with other fluoroquinolone antibiotics including structurally similar ciprofloxacin, while F9 is a general fluoroquinolone binder that likely bound to the antigen at the common pyridone-carboxylic acid pharmacophore. These scFv-based receptors were successfully applied to the development of one-step fluorescent biosensor which can detect fluoroquinolone antibiotics at concentrations below the level suggested in animal drug application guidelines. The strategy described in this report can be applied to developing convenient field biosensors that can qualitatively detect overused/misused antibiotics in the livestock drinking water.     

Correction to: Microhabitats of sharknose goby (Elacatinus evelynae) cleaning stations and their links with cleaning behaviour

Coral Reefs - A correction to this paper has been published: https://doi.org/10.1007/s00338-021-02111-z     

A highly sensitive aptasensor towards Plasmodium lactate dehydrogenase for the diagnosis of malaria

Finding a highly sensitive diagnostic technique for malaria has challenged scientists for the last century. In the present study, we identified versatile single-strand DNA aptamers for Plasmodium lactate dehydrogenase (pLDH), a biomarker for malaria, via the Systematic Evolution of Ligands by EXponential enrichment (SELEX). The pLDH aptamers selectively bound to the target proteins with high sensitivity (K(d)=16.8-49.6 nM). The selected aptamers were characterized using an electrophoretic mobility shift assay, a quartz crystal microbalance, a fluorescence assay, and circular dichroism spectroscopy. We also designed a simple aptasensor using electrochemical impedance spectroscopy; both Plasmodium vivax LDH and Plasmodium falciparum LDH were selectively detected with a detection limit of 1 pM. Furthermore, the pLDH aptasensor clearly distinguished between malaria-positive blood samples of two major species (P. vivax and P. falciparum) and a negative control, indicating that it may be a useful tool for the diagnosis, monitoring, and surveillance of malaria.     

Protein folding as posttranslational regulation: evolution of a mechanism for controlled plasma membrane expression of a G protein-coupled receptor

Recent studies reveal that a number of G protein-coupled receptors (GPCRs) and other proteins are expressed inefficiently at the site normally associated with their biological action. In the case of some GPCRs, large amounts of receptor (perhaps more than half) may be destroyed without ever binding ligand or even arriving at the plasma membrane. For the human GnRH receptor (GnRHR), this apparent inefficiency has evolved under strong and convergent evolutionary pressure. The result is a human GnRHR molecule that is delicately balanced between either expression at the plasma membrane (PM) or retention/degradation in the endoplasmic reticulum, an effect mediated by engagement with the cellular quality control system. This balance appears to be the reason that the human receptor, but not the rat or mouse counterpart (which are more robustly routed to the PM), is highly susceptible to single-point mutations that result in disease. A single change in net charge is sufficient to tip the balance in favor of the endoplasmic reticulum and diminish GnRHR available at the PM. The apparent paradox that results from observing convergent pressure for evolution of a receptor that is both inefficiently produced and highly susceptible to mutational disease suggests that this approach must offer a strong advantage. This review focuses on the evolved mechanisms and considers that this is an underappreciated mechanism by which the cell controls functional levels of receptors and other proteins at the posttranslational level.     

Acute and chronic electroconvulsive shock in rats: effects on peripheral markers of neuronal injury and glial activity

Electroconvulsive therapy is considered one of the most effective treatments of major depression, but controversy still exists on whether it may be brain damaging. The aim of this work was to evaluate the cerebrospinal fluid (CSF) levels of neuron specific enolase (NSE), protein S100B and lactate of rats submitted to acute and chronic models of ECS. Rats were submitted to either one shock (acute) or a series of eight shocks, applied one at every 48 h (chronic). CSF samples were collected at 0, 3, 6, 12, 24, 48 and 72 h after the shock in the acute model and at these same time intervals after the last shock in the chronic model. Both models did not produce significant alterations in the levels of NSE. S100B levels were significantly increased at 6 h in the chronic model (p<0.0001). There was a significant increase in the levels of lactate at 0 h in both models (p<0.001). These results support the proposition that ECS does not produce neural damage, and suggest that the alterations in the levels of S100B and lactate may reflect an astrocytic activity of a protective nature.     

Genotoxicity of diphenyl diselenide in bacteria and yeast

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. This may increase the risk of human exposure to the chemical at the workplace. We have determined its mutagenic potential in the Salmonella/microsome assay and used the yeast Saccharomyces cerevisiae to assay for putative genotoxicity, recombinogenicity and to determine whether DNA damage produced by DPDS is repairable. Only in exponentially growing cultures was DPDS able to induce frameshift mutations in S. typhimurium and haploid yeast and to increase crossing over and gene conversion frequencies in diploid strains of S. cerevisiae. Thus, DPDS presents a behavior similar to that of an intercalating agent. Mutants defective in excision-resynthesis repair (rad3, rad1), in error-prone repair (rad6) and in recombinational repair (rad52) showed higher than WT-sensitivity to DPDS. It appears that this compound is capable of inducing single and/or double strand breaks in DNA. An epistatic interaction was shown between rad3-e5 and rad52-1 mutant alleles, indicating that excision-resynthesis and strand-break repair may possess common steps in the repair of DNA damage induced by DPDS. DPDS was able to enhance the mutagenesis induced by oxidative mutagens in bacteria. N-acetylcysteine, a glutathione biosynthesis precursor, prevented mutagenesis induced by DPDS in yeast. We have shown that DPDS is a weak mutagen which probably generates DNA strand breaks through both its intercalating action and pro-oxidant effect.