Evaluation of different strategies for the development of amperometric biosensors for L-lactate

Two strategies were investigated for the development of lactate biosensors based on sol-gel matrixes and polysulfone composite films, both containing L-lactate dehydrogenase (LDH). Firstly, reagentless disposable screen-printed electrodes (SPE's) with Meldola's Blue (MB) and the cofactor NAD(+) inside a sol-gel matrix were prepared. These showed relatively low sensitivities (260 microA/M). Secondly, mediator-modified-polysulfone-graphite composite films deposited over both cylindrical epoxy-graphite and SPE's. These electrodes showed enhanced performance characteristics: improved sensitivity (80 mA/M), detection limit (0.87 microM) and reproducibility (2%). Reagentless electrodes, incorporating NAD(+) in the polysulfone film, had a decreased sensitivity, although better than that achieved by the sol-gel electrodes. While sol-gel electrodes showed a linear range between 1.25 x 10(-4) and 2.48 x 10(-3)M, the epoxy-graphite composite electrodes based on polysulfone composite films allowed the detection of lactate at a linear range of lower concentrations from 1 x 10(-6) to 1.2 x 10(-5)M. Finally, the performance of the LDH-MB-polysulfone-composite film-based SPE's in a flow system was studied. Short response times were obtained (t<30s). Furthermore, repeatability and reproducibility values were notably improved, especially when working with electrodes covered with a polyamide layer prepared with N-(2-aminoethyl)-piperazine.     

Scientific rationale for the development of gene therapy strategies for Parkinson's disease

The ever-evolving understanding of the neuronal systems involved in Parkinson's disease together with the recent advances in recombinant viral vector technology has led to the development of several gene therapy applications that are now entering into clinical testing phase. To date, four fundamentally different approaches have been pursued utilizing recombinant adeno-associated virus and lentiviruses as vectors for delivery. These strategies aim either to restore the lost brain functions by substitution of enzymes critical for synthesis of neurotransmitters or neurotrophic factors as a means to boost the function of remaining neurons in the diseased brain. In this review we discuss the differences in mechanism of action and describe the scientific rationale behind the currently tested gene therapy approaches for Parkinson's disease in some detail and pinpoint their individual unique strengths and weaknesses.     

Evaluation of industrial yeasts for pathogenicity.

Eleven yeasts representative of species of industrial interest were compared with Candida albicans for their potential pathogenicity for untreated and cortisone-treated mice. Only C. tropicalis produced a progressive infection similar to that produced by C. albicans. Candida lipolytica, Torulopsis spp., and Hansenula polymorpha were not recovered from mice 6 days after inoculation. Kluyveromyces fragilis, C. pseudotropicalis, C. utilis, C. guilliermondii and C. maltosa were recovered from mice but did not produce evidence of infection.     

Oligo/polynucleotide-based gene modification: strategies and therapeutic potential

Oligonucleotide- and polynucleotide-based gene modification strategies were developed as an alternative to transgene-based and classical gene targeting-based gene therapy approaches for treatment of genetic disorders. Unlike the transgene-based strategies, oligo/polynucleotide gene targeting approaches maintain gene integrity and the relationship between the protein coding and gene-specific regulatory sequences. Oligo/polynucleotide-based gene modification also has several advantages over classical vector-based homologous recombination approaches. These include essentially complete homology to the target sequence and the potential to rapidly engineer patient-specific oligo/polynucleotide gene modification reagents. Several oligo/polynucleotide-based approaches have been shown to successfully mediate sequence-specific modification of genomic DNA in mammalian cells. The strategies involve the use of polynucleotide small DNA fragments, triplex-forming oligonucleotides, and single-stranded oligodeoxynucleotides to mediate homologous exchange. The primary focus of this review will be on the mechanistic aspects of the small fragment homologous replacement, triplex-forming oligonucleotide-mediated, and single-stranded oligodeoxynucleotide-mediated gene modification strategies as it relates to their therapeutic potential.     

Molecular breeding strategies for the modification of lipid composition

Summary Lipids are key components of all living cells. Acyl lipids and sterols provide the matrix of the biological membranes that both define the boundaries of cells and organelles, and act as sites for the trafficking of molecules within and into/out of cells. Lipids are also important metabolic intermediates and the most efficient form of energy storage that is available to a cell. It is the latter, energy-storing function that is of most relevance to this review. Storage lipids are accumulated in abundance in many of our most important crops, including maize, soybean, rapeseed, and oil palm, giving rise to a commerical sector valued at over $50 billion/year. Because the storage lipids of the major global oil crops have a relatively restricted composition, there is great interest in using all available breeding technologies, whether traditional or modern, to enhance the variation in lipid quality in existing crops and/or to domesticate new crops that already accumulate useful novel lipids. Over the past few decades, there has been a great deal of effort to manipulate fatty acid composition in order to produce novel lipids, especially for industrial applications. However, these attempts, many based on genetic engineering, have met with only limited commercial success-to date. More recently, there has been a resurgence of interest in the modification of both acyl and non-acyl lipids to enhance the nutritional quality of plant oils. In this review, we will examine the background to plant lipid modification and some of the latest developments, with a particular focus on edible oils.     

Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides

Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogeneous materials that could have improved biological properties. We describe for the first time chemical reduction and oxidation methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purification with hydrophobic interaction chromatography followed by analysis using reversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chain disulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60-90%, allowing for control of the distribution of molecular species. The resulting conjugates are highly active both in vitro and in vivo and are well tolerated at efficacious doses.     

Evaluation of the MicroScan enzyme-based system for the identification of foodborne yeasts

Eighty-nine strains representing 36 species of foodborne yeasts isolated from fruit juice concentrates were identified using the Baxter MicroScan enzyme-based kit, conventional tests according to a simplified identification method (SIM), and the API 20C kit. Of the 15 test species included in the MicroScan database, only 40% were correctly identified; 13% gave scores of unacceptably low probabilities, 20% were misidentified, and 27% could not be identified. Of the 21 test species not in the MicroScan database, 38% were misidentified and 62% produced biocodes with between-species differences not larger than differences between strains within species. The reliability of the MicroScan enzyme-based system is questioned, in that different results were sometimes obtained upon retesting the same strains. The MicroScan enzyme-based system is rapid, providing results within 4 h. However, because of its restricted and specific database and unreliability, the system appears to be unsuited for the identification of foodborne yeasts.     

Evaluation of the Biolog system for the identification of food and beverage yeasts

The inconvenience of conventional yeast identification methods has resulted in the development of rapid, commercial systems, mainly for clinical yeast species. The Biolog system (Biolog Inc., Hayward, CA, USA) is a new semi-automated, computer-linked technology for rapid identification of clinical and non-clinical yeasts. The system is based around a microtitre tray and includes assimilation and oxidation tests. This paper evaluates the Biolog system for the identification of 21 species (72 strains) of yeasts of food and wine origin. Species correctly identified included Saccharomyces cerevisiae , Debaryomyces hansenii , Yarrowia lipolytica , Kluyveromyces marxianus , Kloeckera apiculata , Dekkera bruxellensis and Schizosaccharomyces pombe. Zygosaccharomyces bailii and Zygosaccharomyces rouxii were identified correctly 50% of the time and Pichia membranaefaciens 20% of the time.     

Contemporary gene targeting strategies for the novice

Gene targeting in mouse embryonic stem (ES) cells is a fundamental methodology for generating mice with precise genetic modifications. Although there are many complex gene targeting strategies for creating a variety of diverse mutations in mice, most investigators initially choose to generate a null allele. Here we provide a guide for the novice to generate a null allele for a protein coding gene using a fundamental gene targeting strategy. Ultimately, a well considered gene targeting strategy saves significant amounts of time, money, and research animal lives. The straightforward strategy presented here bypasses many of the pitfalls associated with gene knockouts generated by novices. This guide also serves as a foundation for subsequently designing more complex gene targeting strategies.     

Targeted gene modification for gene therapy of stem cells

Ideally, gene therapy would correct the specific gene defect without adding potentially harmful extraneous DNA sequences. Such correction can be obtained with homologous recombination between input DNA sequences and identical (homologous) sequences in the genomic target gene. The development of techniques for obtaining virtually pure populations of hematopoietic stem cells should permit the use of the highly efficient nuclear microinjection methods for transfer of DNA. These techniques combined with new highly sensitive methods for detecting cells with the specified genetic modification of nonexpressed genes would make homologous recombination-mediated gene therapy feasible for hematopoietic stem cells. These advances are reviewed with particular emphasis on approaches to targeted gene modification of hematopoietic stem cells and speculation on directions for future research.     

Evaluation of the Pro-Lab ID ring system for the identification of medically important yeasts

We evaluated 151 coded isolates of medically important yeast species belonging to the genera Candida, Cryptococcus, Geotrichum, Rhodoturula, Saccharomyces and Torulopsis using the newly developed rapid Pro-Lab Identification Ring, PL 960 system (PLID-Ring). All isolates were concurrently identified by the API 20C and conventional procedures comprising macro- and micromorphology, assimilation and fermentation of various carbon and nitrogen compounds. The PLID-Ring system identified isolates of Candida albicans, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, Rhodotorula rubra, and Torulopsis glabrata with 100% accuracy in 24 h. This system identified C guilliermondii and S. cerevisiae isolates with an accuracy of 90% and 86%, respectively, while those belonging to Cr. neoformans, T. candida (= C. famata), C. rugosa and C. tropicalis were identified with 38.4%, 50%, 12.5% and 50% accuracy, respectively. Three isolates of Cr. laurentii were not identified by the PLID-Ring system. The overall accuracy of the PLID-Ring system was 81.45% (123 of 151 isolates). However, the system does not include species such as Cr. laurentii in its data base. When these three Cr. laurentii isolates were excluded from the evaluation, the accuracy of the PLID-Ring system increased from 81.45% to 83.1%.     

Development of resources for the analysis of gene function in Pucciniomycotina red yeasts

The Pucciniomycotina is an important subphylum of basidiomycete fungi but with limited tools to analyze gene functions. Transformation protocols were established for a Sporobolomyces species (strain IAM 13481), the first Pucciniomycotina species with a completed draft genome sequence, to enable assessment of gene function through phenotypic characterization of mutant strains. Transformation markers were the URA3 and URA5 genes that enable selection and counter-selection based on uracil auxotrophy and resistance to 5-fluoroorotic acid. The wild type copies of these genes were cloned into plasmids that were used for transformation of Sporobolomyces sp. by both biolistic and Agrobacterium-mediated approaches. These resources have been deposited to be available from the Fungal Genetics Stock Center. To show that these techniques could be used to elucidate gene functions, the LEU1 gene was targeted for specific homologous replacement, and also demonstrating that this gene is required for the biosynthesis of leucine in basidiomycete fungi. T-DNA insertional mutants were isolated and further characterized, revealing insertions in genes that encode the homologs of Chs7, Erg3, Kre6, Kex1, Pik1, Sad1, Ssu1 and Tlg1. Phenotypic analysis of these mutants reveals both conserved and divergent functions compared with other fungi. Some of these strains exhibit reduced resistance to detergents, the antifungal agent fluconazole or sodium sulfite, or lower recovery from heat stress. While there are current experimental limitations for Sporobolomyces sp. such as the lack of Mendelian genetics for conventional mating, these findings demonstrate the facile nature of at least one Pucciniomycotina species for genetic manipulation and the potential to develop these organisms into new models for understanding gene function and evolution in the fungi.     

Evaluation of selective media for enumeration of yeasts during wine fermentation

The growth of individual species of yeasts during wine fermentations was measured by plating wine samples on malt extract, ethanol sulphite and lysine agars. Colonies of Saccharomyces cerevisiae dominated on plates of malt extract agar and sometimes masked the presence of other non- Saccharomyces species. Lysine agar suppressed the growth of S. cerevisiae and enabled the enumeration of non- Saccharomyces species such as Kloeckera apiculata, Candida stellata and Saccharomycodes ludwigii. The growth of non- Saccharomyces yeasts on ethanol sulphite agar was variable.     

Selection strategies for the development of rye introgression libraries

Computer simulations can be employed to find optimal procedures for developing introgression libraries in rye with marker-assisted backcrossing. Our objectives were to investigate the effects of the employed (1) breeding scheme, (2) selection strategy, and (3) population sizes on the donor genome coverage of the library, the number of introgression lines carrying additional donor chromosome segments outside the target regions, and the number of required marker data points. With respect to these target criteria, a BC₃S₂ breeding scheme and increasing population sizes from early to advanced generations were superior to a BC₂S₃ breeding scheme and constant population sizes. The smallest number of donor segments outside the target regions was reached with a three-stage selection strategy, which consists on selection for the target segment, selection for recombination at flanking markers and selection for recurrent parent alleles across the entire genome. Omitting the selection for flanking markers in generation BC₁ reduced considerably the number of required marker data points. A pre-selection of chromosomes consisting completely of donor genome in BC₁ was advantageous, if the effort in the breeding nursery should kept minimum. Adopting the described designs can help rye breeders to successfully develop introgression libraries.     

Genetic control of the modification of the radiosensitivity of yeasts by oxygen and hypoxic sensitizers

Diploid cells of the wild-type yeast Saccharomyces cerevisiae, mutants homozygous with respect to rad2 and rad54 loci as well as a double mutant with both these loci in homozygous state were used to demonstrate the previously observed (in other yeast strains) genetic determination of radiosensitivity modification of hypoxic cells by oxygen and electron-affinic compounds. It was shown that both oxygen effect and the effect of hypoxic sensitizers depended on the activity of repair systems. A possible mechanism of participation of postradiation recovery in modification of yeast cell radiosensitivity is discussed.     

New strategies for cardiovascular gene therapy

Cardiovascular diseases are among the major targets for gene therapy. Initially, clinical experiments of gene transfer of vascular endothelial growth factor (VEGF) improved vascularization and prevented the amputation in patients with critical leg ischemia. However, the majority of trials did not provide conclusive results and therefore further preclinical studies are required. Importantly, data indicate the necessity of regulated expression of angiogenic factors, particularly VEGF, to obtain the therapeutic effect. It is also suggested that the combined delivery of two or more genes may improve the formation of mature vasculature and therefore may be more effective in the amelioration of ischemia. Moreover, experimental approaches in animal models displayed the promise of gene transfer modulating the inflammatory processes and oxidant status of the cells. Particularly, the concept of preemptive gene therapy has been tested, and recent studies have demonstrated that overexpression of heme oxygenase-1 or extracellular superoxide dismutase can prevent heart injury by myocardial infarction induced several weeks after gene instillation. The combination of a preemptive strategy with regulated gene expression, using the vectors in which the therapeutic transgene is driven by exogenously or endogenously controllable promoter, offers another modality. However, we hypothesize that regulatable gene therapy, dependent on the activity of endogenous factors, might be prone to limitations owing to the potential disturbance in the expression of endogenous genes. Here, we demonstrated some indications of these drawbacks. Therefore, the final acceptance of these promising strategies for clinical trials requires careful validation in animal experiments.     

Surviving in the presence of sulphur dioxide: strategies developed by wine yeasts

Sulphur dioxide has been used as a common preservative in wine since at least the nineteenth century. Its use has even become essential to the making of quality wines because of its antioxidant, antioxidasic and antiseptic properties. The chemistry of SO₂ in wine is fairly complex due to its dissociation into different species and its binding to other compounds produced by yeasts and bacteria during fermentation. The only antiseptic species is the minute part remaining as molecular SO₂. The latter concentration is both dependent on pH and concentration of free bisulphite. However, certain yeast species have developed cellular and molecular mechanisms as a response to SO₂ exposure. Some of these mechanisms are fairly complex and have only been investigated recently, at least for the molecular mechanisms. They include sulphite reduction, sulphite oxidation, acetaldehyde production, sulphite efflux and the entry into viable but not culturable state, as the ultimate response. In this review, the chemistry of SO₂ in wine is explained together with the impact of SO₂ on yeast cells. The different defence mechanisms are described and discussed, mostly based on current knowledge available for Saccharomyces cerevisiae.