Carlos A. Stella

Study on fatty acid binding by proteins in yeast. Dissimilar results in Saccharomyces cerevisiae and Yarrowia lipolytica

1. The presence of soluble proteins with fatty acid binding activity was investigated in cell-free extracts from Saccharomyces cerevisiae and Yarrowia lipolytica cultures. 2. No significant fatty acid binding by proteins was detected in S. cerevisiae, even when grown on a fatty acid-rich medium, thus indicating that such proteins are not essential to fatty acid metabolism. 3. An inducible fatty acid binding protein (K0.5 = 3-4 microM) was found in Y. lipolytica which had grown on a minimal medium with palmitate as the sole source of carbon and energy. 4. The relative molecular mass of this protein was 100,000 as inferred from Sephacryl S-200 gel filtration.

RAS2/PKA pathway activity is involved in the nitrogen regulation of L-leucine uptake in Saccharomyces cerevisiae.

The aim of the present work is to study the participation of RAS2/PKA signal pathway in the nitrogen regulation of L-leucine transport in yeast cells. The study was performed on Saccharomyces cerevisiae isogenic strains with the normal RAS2 gene, the RAS2val19 mutant and the disrupted ras2::LEU2. These strains bring about different activities of the RAS2/PKA signal pathway, L-(14C)-Amino acid uptake measurements were determined in cells grown in a rich YPD medium with a mixed nitrogen source or in minimal media containing NH4+ or L-proline as the sole nitrogen source. We report herein that in all strains used, even in those grown in a minimal proline medium, the activity of the general amino acid permease (GAP1) was not detected. L-Leucine uptake in these strains is mediated by two kinetically characterized transport systems. Their KT values are of the same order as those of S1 and S2 L-leucine permeases. Mutation in the RAS2 gene alters initial velocities and Jmax values in both high and low affinity L-leucine transport systems. Activation of the RAS2/PKA signalling pathway by the RAS2val19 mutation, blocks the response to a poor nitrogen source whereas inactivation of RAS2 by gene disruption, results in an increase of the same response.

A Simple Chemical Method for Rendering Wild-Type Yeast Permeable to Brefeldin A That Does Not Require the Presence of an erg6 Mutation

The present work aims to develop a growth medium to render a wild-type strain of Saccharomyces cerevisiae permeable to the antifungal drug Brefeldin A. In the current study, a synthetic medium containing 0.1% L-proline and supplemented with 3.0 × 10-3% SDS is employed. When Brefeldin A is added to this medium, a wild-type strain shows increased growth sensitivity and a diminished transport of the amino acid L-leucine. Since Brefeldin A exerts its effect on the endoplasmic reticulum and the Golgi apparatus, the medium permits the study of the drug effect on the intracellular traffic of L-leucine permeases.

Inhibitory action of palmitic acid on the growth ofSaccharomyces cerevisiae

High concentrations of long-chain fatty acids have been found to be harmful to mammalian cells and prokaryotic organisms. This effect was investigated in Saccharomyces cerevisiae. Addition of 3 mmol/L palmitate to a yeast extract-peptone medium caused a significant inhibition of cell growth during the first 2 d of incubation, followed by renewed growth and palmitate utilization. Inhibition was also observed with palmitate concentrations down to 0.1 mmol/L. As inferred from catalase activity determinations, this effect was found to correlate with the absence of peroxisome proliferation. Finally, no inhibition was observed in exponential-phase cultures or in the presence of 0.1 g/L glucose, this suggesting that the physiological state of the cell may determine whether its growth will be inhibited by fatty acids.

L-Phenylalanine Transport in Saccharomyces cerevisiae: Participation of GAP1, BAP2, and AGP1

We focused on the participation of GAP1, BAP2, and AGP1 in L-phenylalanine transport in yeast. In order to study the physiological functions of GAP1, BAP2, and AGP1 in L-phenylalanine transport, we examined the kinetics, substrate specificity, and regulation of these systems, employing isogenic haploid strains with the respective genes disrupted individually and in combination. During the characterization of phenylalanine transport, we noted important regulatory phenomena associated with these systems. Our results show that Agp1p is the major transporter of the phenylalanine in a gap1 strain growing in synthetic media with leucine present as an inducer. In a wild type strain grown in the presence of leucine, when ammonium ion was the nitrogen source, Bap2p is the principal phenylalanine carrier.

Effect of Potassium on Saccharomyces cerevisiae Resistance to Fluconazole

ABSTRACT Susceptibility of strain S288c of Saccharomyces cerevisiae to fluconazole was assayed in the presence and absence of KCl. Addition of 150 mM KCl renders the strain more sensitive to the antifungal agent. The effect is caused by the K+ ion rather than the anion or the osmolarity of the medium. The increase in sensitivity does not modify the values of intracellular and extracellular pH established in the presence of KCl.

Biochemical study of idebenone effect on mitochondrial metabolism of yeast

The aim of this work was to study the effect of the drug idebenone on the growth of a strain of Saccharomyces cerevisiae yeast and its respiratory‐deficient mutant (rho0). We took this yeast as a model system of the interaction of the drug with mammalian cells. The effect of idebenone was evaluated in rich and minimal media. In the S288c strain, idebenone exerted a growth inhibitory effect in concentrations higher than 50 μM in media containing a carbon source consumed at mitochondrial level. In conditions of low oxygen supply, idebenone allows yeast to keep a cellular yielding comparable with conditions of normal oxygen supply. Also, the presence of idebenone in the growth media increased by 50% the fluorescence signal of rhodamine 123, indicating a higher mitochondrial membrane potential. The results could explain the effect of idebenone in the treatment of diseases in which oxygen deficiency alters the energetic metabolism of the cell.

Student assessment in large‐enrollment biology classes

Enrollments into first‐year university biology courses may be very large, and therefore evaluating student learning can represent quite a challenge. In this article, we present our experience in assessing students by means of an assessment instrument called “Understand Before Choosing” (UBC). It has been used for six semesters, and its performance has been compared with two other common means of assessment, the use of multiple‐choice questions and the use of open‐ended questions. UBC consists of a text (100 lines, nearly 700 words) on the subject being tested, and a set of carefully worded questions that require the selection of one of five crafted options to be answered. To choose the best option, a student needs to understand the concepts embedded in the text.

Teaching lactose metabolism

We developed an experimental didactic proposal to teach both carbohydrate metabolism and lactose intolerance as the disease related to that metabolism. Therefore, we implemented an empirical strategy consisting of inexpensive and nontoxic components for which students do not need to know any of the laboratory techniques. The fact that students were able to discuss their own results obtained from the experiments performed in their classroom gave them an additional motivation to learn the subject.

L-proline as a nitrogen source increases the susceptibility of Saccharomyces cerevisiae S288c to fluconazole.

Fluconazole inhibition ofSaccharomyces cerevisiae S288c growth was evaluated in media containing ammonia,l-proline orl-leucine as a nitrogen source. Growth inhibition by fluconazole was maximum whenl-proline was used as a nitrogen source, while rhodamine 6G accumulation and fluconazole resistance were the highest when ammonia was the sole nitrogen source.