The encapsulation of living cells in materials with good optical and mechanical properties often produces death or stress due to the release of toxic byproducts originated during the synthesis. We present here a method to assess the cellular stress that silica entrapment exerts over living cells taking into account the main preparation variables such as the nature of the silica source, protecting functional groups, total solid concentration, or indirect procedures. Measurement of the cellular stress status of genetically modified Sacharomyces cereVisiae, a true biological probe, allowed us to perform a quantitative analysis of cellular stress in a short time basis (compared to conventional long-term viability tests), opening the gate for a more sophisticated approach to optimize the synthesis conditions. In addition, the aforementioned findings allowed the preparation of novel materials with enhanced optical and mechanical properties. The relation of cellular stress with initial viability is also discussed.

The Saccharomyces cerevisiae UGA4 gene encodes a permease capable of importing ␥-aminobutyric acid (GABA) and ␦-aminolevulinic acid (ALA) into the cell. GABA-dependent induction of this permease requires at least two positive-acting proteins, the specific factor Uga3 and the pleiotropic factor Uga35/Dal81. UGA4 is subjected to a very complex regulation, and its induction is affected by the presence of extracellular amino acids; this effect is mediated by the plasma membrane amino acid sensor SPS. Our results show that leucine affects UGA4 induction and that the SPS sensor and the downstream effectors Stp1 and Stp2 participate in this regulation. Moreover, we found that the Uga3 and Uga35/Dal81 transcription factors bind to the UGA4 promoter in a GABA-dependent manner and that this binding is impaired by the presence of leucine. We also found that the Leu3 transcription factor negatively regulates UGA4 transcription, although this seems to be through an indirect mechanism.

d-aminolevulinic acid, the precursor of porphyrin biosynthesis has been used to induce the endogenous synthesis of the photosensitiser protoporphyrin IX for photodynamic therapy in the treatment of various tumours. The aim of this work was to characterise the d-aminolevulinic acid transport system in the murine mammary adenocarcinoma cell line LM3 using 14 C-daminolevulinic acid, to finally improve d-aminolevulinic acid incorporation in mammalian cells. Our results showed that daminolevulinic acid is incorporated into these cells by two different mechanisms, passive diffusion which is important at the beginning of the incubation, and active transport. Specificity assays suggested that the transporter involved in d-aminolevulinic acid incorporation is a BETA transporter, probably GAT-2.

No abstract

c-Aminobutyric acid (GABA) transport and catabolism in Saccharomyces cerevisiae are subject to a complex transcriptional control that depends on the nutritional status of the cells. The expression of the genes that form the UGA regulon is inducible by GABA and sensitive to nitrogen catabolite repression (NCR). GABA induction of these genes is mediated by Uga3 and Dal81 transcription factors, whereas GATA factors are responsible for NCR. Here, we show how members of the UGA regulon share the activation mechanism. Our results show that both Uga3 and Dal81 interact with UGA genes in a GABA-dependent manner, and that they depend on each other for the interaction with their target promoters and the transcriptional activation. The typical DNA-binding domain Zn(II) 2 -Cys 6 of Dal81 is unnecessary for its activity and Uga3 acts as a bridge between Dal81 and DNA. Both the trans-activation activity of the GATA factor Gln3 and the repressive activity of the GATA factor Dal80 are exerted by their interaction with UGA promoters in response to GABA, indicating that Uga3, Dal81, Gln3 and Dal80 all act in concert to induce the expression of UGA genes. So, an interplay between the factors responsible for GABA induction and those responsible for NCR in the regulation of the UGA genes is proposed here.

The Saccharomyces cerevisiae UGA4 gene, which encodes the c-aminobutyric acid (GABA) and d-aminolaevulinic acid (ALA) permease, is well known to be regulated by the nitrogen source. Its expression levels are low in the presence of a rich nitrogen source but are higher when a poor nitrogen source is used. In addition, GABA can induce UGA4 expression when cells are grown with proline but not when they are grown with ammonium. Although vast amounts of evidence have been gathered about UGA4 regulation by nitrogen, little is known about its regulation by the carbon source. Using glucose and acetate as rich and poor carbon source respectively, this work aimed to shed light on hitherto unclear aspects of the regulation of this gene. In poor nitrogen conditions, cells grown with acetate were found to have higher UGA4 basal expression levels than those grown with glucose, and did not show UGA4 induction in response to GABA. Analysis of the expression and subcellular localization of the transcription factors that regulate UGA4 as well as partial deletions and site-directed mutations of the UGA4 promoter region suggested that there are two parallel pathways that act in regulating this gene by the carbon source. Furthermore, the results demonstrate the existence of a new factor operating in UGA4 regulation.

d-aminolevulinic acid (ALA) is the precursor in the biosynthesis of porphyrins. The knowledge of both the regulation of ALA entrance and efflux from the cells and the control of porphyrin biosynthesis is essential to improve ALA-mediated photodynamic therapy. In this work, we studied the regulation of ALA uptake and efflux by endogenously accumulated ALA and/or porphyrins in murine mammary adenocarcinoma cells. Under our set of conditions, the haem synthesis inhibitor succinyl acetone completely prevented porphobilinogen and porphyrin synthesis from ALA, and led to an increase in the intracellular ALA pool. However, neither intracellular ALA nor porphyrin pools regulate ALA uptake or efflux during the first 15 min of the process. Based on temperature dependence data, ALA but not g-aminobutyric acid (GABA) efflux is mediated by a diffusion mechanism. Moreover, the addition of extracellular GABA not only did not influence the rate of ALA efflux but on the contrary it affected ALA uptake, showing the contribution of a saturable mechanism for the uptake, but not for the efflux of ALA from the cells.

Asr1, a tomato gene induced by abiotic stress, belongs to a family, composed by at least three members, involved in adaptation to dry climates. To understand the mechanism by which proteins of this family seem to protect cells from water loss in plants, we expressed Asr1 in the heterologous expression system Saccharomyces cerevisiae under the control of a galactose-inducible promoter. In a mutant yeast strain deficient in one component of the stressresponsive high-osmolarity glycerol (HOG) pathway, namely the MAP kinase Hog1, the synthesis of ASR1 protein restores growth under osmotic stress conditions such as 0.5 M NaCl and 1.2 M sorbitol. In contrast, the rescuing of this phenotype was less evident using a wild-type strain or the upstream MAP kinase kinase (Pbs2)-deficient strain. In both knock-out strains impaired in glycerol synthesis because of a dysfunctional HOG pathway, but not in wild-type, ASR1 led to the accumulation of endogenous glycerol in an osmotic stress-independent and unrestrained manner. These data suggest that ASR1 complements yeast HOG-deficient phenotypes by inducing downstream components of the HOG pathway. The results are discussed in terms of the function of ASR proteins in planta at the molecular and cellular level.