We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and 15N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose‐repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids β‐oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the post‐transcriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology. ### Synopsis Organisms, varying from unicellular bacteria to mammalian species, rely on the uptake of nutrients from the environment to sustain energy, metabolism and growth. They have therefore evolved numerous strategies to adapt to their changing environment. Such programs involve instantaneous responses (changes in intracellular metabolites, activation/inhibition of enzymes by effectors and of proteins through post‐translational modifications) as well as slower processes that affect the levels of macromolecules (transcription, translation, mRNA and protein degradation). The yeast Saccharomyces cerevisiae can adapt very well to different environments, and is therefore an ideal system to investigate the molecular system response to such changes in the environment. In recent large‐scale transcriptome analyses of S. cerevisiae grown under various nutrient limitations ([Boer et al , 2003][1]; [Daran‐Lapujade et al , 2004][2]; [Saldanha et al , 2004][3]; [Wu et al , 2004b][4]; [Tai et al , 2005][5]), specific transcriptional responses to the limiting nutrient were identified. However, it is not known to what extent these major transcriptional responses are actually translated into quantitatively identical responses at the protein level. With the aim of investigating the proteomic response of S. cerevisiae grown under different nutrient limitation regimes (carbon and nitrogen limitation) and to assess to what extent changes at the protein level are reflected in changes at the transcriptional level, we performed an extensive quantitative proteome analysis of chemostat‐cultivated S. cerevisiae limited for glucose or ammonia ([Figure 1][6]). The yeast strains were therefore labeled in vivo by metabolic stable isotope labeling with 15N, enabling the differential quantification of protein expression levels directly by mass spectrometry ([Ong et al , 2003][7]; [Romijn et al , 2003][8]). The experimental approach that combined isotope labeling with one‐dimensional gel separation of proteins and LC‐MS/MS analysis of peptides generated by tryptic proteolysis of 1D gel bands led to a quantitative data set that appeared to be less biased compared to standard 2D gel‐based analysis, as proteins from different subcellular compartments, including membrane proteins, and proteins with extreme isoelectric points and molecular weights were identified. In order to reduce error and noise in the data, we used rather stringent criteria to filter the protein expression levels, resulting in a data set of 102 proteins that were considered as significantly changed. We also performed a reverse isotope labeling experiment from which we concluded that the reproducibility of the expression ratios was within 20% at least for 3/4 of the quantified proteins, reflecting a good experimental and biological reproducibility. Additionally, we performed multiple LC‐MS/MS analysis, and analyzed our data stringently, leading to the quantification of about 750 proteins, with an average of nearly five peptide pairs per protein. We believe that this is at present one of the best quality quantitative proteomics studies, enabling a proper comparison with transcriptome data. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose‐repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids β‐oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging alternative nitrogen sources and in protein degradation. Our data set also revealed numerous proteins that seem to be present rather exclusively under either nitrogen or carbon limitation. Some of these proteins had poorly described functions, but are likely important for survival under carbon or nitrogen limitation. We also examined whether proteins known to be components of functional and/or structural protein complexes showed differential coexpression. Interestingly, we observed that there is a good correlation in coexpression for various protein complexes, such as the vacuolar H+‐ATPase complex, the cytochrome c oxidase complex and the proteasome. However, for some complexes (e.g. the F0/F1 ATP synthase), opposite regulation of the different subunits was observed, making overall conclusions not straightforward. Our extensive and high‐quality data set enabled us to compare our...

This paper describes a novel fabrication process based on successive wafer-level bonding and releasing steps for stacking several patterned layers of the negative photoresist EPON SU-8. This work uses a polyimide film to enhance previous low temperature bonding technology. The film acts as a temporary substrate where the SU-8 is photopatterned. The poor adhesion between the polyimide film and SU-8 allows the film to be released after the bonding process, even though the film is still strong enough to carry out photolithography. Using this technique, successive adhesive bonding steps can be carried out to obtain complex 3-D multilayer structures. Interconnected channels with smooth vertical sidewalls and freestanding structures are fabricated. Unlike previous works, all the layers are photopatterned before the bonding process yielding sealed cavities and complex three-dimensional structures without using a sacrificial layer. Adding new SU-8 layers reduces the bonding quality because each additional layer decreases the thickness uniformity and increases the polymer crosslinking level. The effect of these parameters is quantified in this paper. This process guarantees compatibility with CMOS electronics and MEMS. Furthermore, the releasing step leaves the input and the output of the microchannels in contact with the outside world, avoiding the usual slow drilling process of a cover. Hence, in addition to the straightforward integration of electrodes on a chip, this fabrication method facilitates the packaging of these microfluidic devices.

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

B cells play an essential role in the immune response. Upon activation they may differentiate into plasma cells that secrete specific antibodies against potentially pathogenic non-self antigens. To identify the cellular proteins that are important for efficient production of these antibodies we set out to study the B cell differentiation process at the proteome level. We performed an in-depth proteomic study to quantify dynamic relative protein expression patterns of several hundreds of proteins at five consecutive time points after lipopolysaccharide-induced activation of B lymphocytes. The proteome analysis was performed using a combination of stable isotope labeling using [¹³C₆]leucine added to the murine B cell cultures, one-dimensional gel electrophoresis, and LC-MS/MS. In this study we identified 1,001 B cell proteins. We were able to quantify the expression levels of a quarter of all identified proteins (i.e. 234) at each of the five different time points. Nearly all proteins revealed changes in expression patterns. The quantitative dataset was further analyzed using an unbiased clustering method. Based on their expression profiles, we grouped the entire set of 234 quantified proteins into a limited number of 12 distinct clusters. Functionally related proteins showed a strong correlation in their temporal expression profiles. The quality of the quantitative data allowed us to even identify subclusters within functionally related classes of proteins such as in the endoplasmic reticulum proteins that are involved in antibody production.

E2F transcription factors control diverse biological processes through regulation of target gene expression. However, the mechanism by which this regulation is established, and the relative contri...