In addition to its other roles, L-serine functions in one-carbon metabolism and is interconvertable with glycine via serine hydroxymethyltransferases. However, the transcriptional response by Saccharomyces cerevisiae to L-serine addition is markedly different from that to glycine, with L-serine acting as a nutrient source rather than one-carbon units. Following addition of excess L-serine, 743 genes showed significant expression changes. Induced functions included amino acid synthesis, some stress responses, and FeS metabolism, while ribosomal RNA processing, ribosome biogenesis and hexose transport were repressed. A co-regulated network of ten transcription factors could together control more than 90% of the induced and repressed genes forming a general response to changes induced by other amino acids or stresses and including the general amino acid control system usually activated in response to starvation for amino acids. A specific response to L-serine was induction of CHA1 encoding serine (threonine) dehydratase. L-serine addition resulted in a substantial transient increase in L-aspartate, which is, rather than L-glutamate, the major metabolite for short-term storage of ammonia derived from degradation of L-serine. L-aspartate synthesis was exclusively through mitochondrial metabolism of L-serine to pyruvate and ammonia, involving Cha1p, cytoplasmic pyruvate carboxylases Pyc1p and Pyc2p, and the cytoplasmic aspartate aminotransferase Aat2p.

The transcriptional activator Gcn4p is considered the master regulator of amino acid metabolism in Saccharomyces cerevisiae and is required for the transcriptional response to amino acid starvation. Here it is shown that Gcn4p plays a previously undescribed role in regulating adaptation to anaerobic growth. A gcn4 mutant exhibited a highly extended lag phase after a shift to anaerobiosis that was the result of L-serine depletion. In addition, the one-carbon metabolism and purine biosynthesis transcriptional regulator Bas1p were strictly required for anaerobic growth on minimal medium, and this was similarly due to L-serine limitation in bas1 mutants. The induction of one-carbon metabolism during anaerobiosis is needed to increase the supply of L-serine from the glycine and threonine pathways. Using a number of experimental approaches, we demonstrate that these transcription regulators play vital roles in regulating L-serine biosynthesis in the face of increased demand during adaptation to anaerobiosis. This increased L-serine requirement is most likely due to anaerobic remodeling of the cell wall, involving de novo synthesis of a large number of very serine-rich mannoproteins and an increase in the total serine content of the cell wall. During anaerobic starvation for L-serine, this essential amino acid is preferentially directed to the cell wall, indicating the existence of a regulatory mechanism to balance competing cellular demands.Saccharomyces cerevisiae can grow rapidly aerobically and anaerobically, and this has led to its use in the study of oxygen sensing and the requirement of molecular oxygen for metabolism. Under anaerobic conditions cells cannot synthesize sterols and unsaturated fatty acids because the two pathways require molecular oxygen (1). Several studies have identified the genome-wide transcriptional responses of yeast growing in aerobic or anaerobic conditions (2-6), however, many environments are subject to dynamic fluctuations in oxygen tension and, hence, there is current interest in how organisms respond to changes in oxygen level (3,7,8). Under anaerobic conditions proteins involved in amino acid metabolism are synthesized at higher levels (9). We have previously reported that cells lacking the Gcn4p transcription factor regulating genes involved in amino acid metabolism have a growth defect under anaerobic conditions. This indicates that there may be an altered requirement for some Gcn4p-dependent aspect of amino acid biosynthesis in anaerobic cells compared with aerobic (10).In S. cerevisiae and related fungi, including Candida albicans, Gcn4p is a master transcriptional activator that directly activates over 30 amino acid biosynthetic genes for biosynthesis of 19 amino acids, as well as regulating many other cellular processes, including purine biosynthesis, organelle biosynthesis, autophagy, glycogen homeostasis, and stress responses (11). The mechanisms involved in Gcn4p regulation have been reviewed by Hinnebusch (12). When cells are subjected to stresses such as amino acid, puri...

In the current study a technique for microencapsulation of human breast adenocarcinoma cells MCF-7 in alginate-chitosan microcapsules is used. Microencapsulation is proposed to generate multicellular tumor spheroids (MTS) based on these cells and to test them further as an in vitro model for anti-tumor drug screening. Cytotoxicity of methotrexate (MTX) was studied on the obtained MTS. A set of MTS with mean size of 150, 200 and 300 m was prepared in function of a cultivation time. After incubation of MTS in cultivation medium containing MTX at concentrations of 1, 2, 10, 50 and 100 nM for 48 hs cell viability was evaluated. MTS were shown to be more resistant to MTX than the monolayer culture, and the resistance to MTX was increased with enhancing a spheroid size. At MTX concentration of 100 nM a number of viable cells in MTS with the size of 300 m was 2.5-fold bigger than that one in monolayer culture. It is suggested that the cells in microencapsulated MTS can better mimic cell behavior in a small size solid tumor than the cells in a monolayer culture. In future microencapsulated MTS can be proposed as a novel in vitro model for anticancer drug screening.