The regulation of MHC class II gene expression controls T-cell activation and, hence, the immune response. Among the nuclear factors observed to bind to conserved DNA sequences in human leukocyte antigen (HLA) class II gene promoters, RFX is of special interest: Its binding is defective in congenital HLA class II deficiency, a disease of class II gene regulation. The cloning of an RFX cDNA has allowed us to show by transfection of a plasmid directing the synthesis of antisense RFX RNA that RFX is a class II gene regulatory factor. RFX is a novel 979-amino-acid DNA-binding protein that contains three structurally and functionally separate domains. The 91-amino-acid DNA-binding domain is distinct from other known DNA-binding motifs but may be distantly related to the helix-loop-helix motif. The most striking property of RFX is that it can bind stably to the class II X box as either a monomer or a homodimer and that the domain responsible for dimerization is distant from and functionally independent of the DNA-binding domain. This distinguishes RFX from other known dimeric DNA-binding proteins. It also implies that an RFX homodimer has two potential DNA-binding sites. We therefore speculate that RFX could form a DNA loop by cross-linking the two X-box sequences found far apart upstream of MHC class II genes.

Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in such proteins is a major challenge in proteomics. In the present study we evaluate the use of enzymatic de-phosphorylation in combination with differential peptide mass mapping for identification of phosphorylated peptides in peptide mixtures derived from in-gel digested phospho-proteins. Phospho-peptides could be identified provided that improved sample preparation methods prior to mass spectrometric analysis were used. An attempt to identify the proteins visualized by [32P] autoradiography in a proteomics study and their phosphorylation sites, demonstrated that protein identification was possible whereas reliable identification of the phospho-peptides requires more protein than normally available in our proteomics studies.

Chromatin is a highly dynamic structure that must respond to different stimuli in order to orchestrate all DNA-dependent processes. Post translational modifications (PTMs) 1 of histones play a major role in regulation of chromatin functionality. Evidence is emerging that not only "classical" histone PTMs, such as methylation, acetylation, and phosphorylation at distinct residues, but also proteolytic processing of nucleosome proteins, known as "histone clipping," can be involved in regulation of key cellular processes, such as transcriptional regulation, cell differentiation, and senescence (1-7).Clipping of the histone H3 N-terminal tail was reported to be associated with gene activation in yeast. Santos-Rosa et al. demonstrated a serine protease activity in S. cerevisiae that cleaves histone H3 after residue Ala21 (A21) during sporulation and stationary phase (1). H3 clipping took place specifically within the promoters of sporulation-induced genes following the induction of transcription and prior to histone eviction from these DNA regions. Prevention of H3 N-tail cleavage by amino acid substitution at the endoproteinase recognition site (H3 Q19A, L20A) abolished expression of these genes, indicating that H3 clipping is essential for productive transcription.The biological significance of histone clipping in higher eukaryotes is not yet understood but also appears to be related to functional commitment by the cell. Duncan et al. demonstrated that histone H3 is proteolytically cleaved by the enzyme Cathepsin L1 (CTSL1) at several sites between residues A21 and S28 during mouse ESC differentiation (5). The "in vitro" proteolytic activity of CTSL1 was found to be dependent on the H3 N-tail PTM status. H3K27me2 increased From the ‡Department

The rising number of proteome projects leads to new challenges for two-dimensional electrophoresis with immobilized pH gradients and different applications of this technique. Not only wide pH gradients such as 4-12 or 3-12 (Görg et al., Electrophoresis 1999, 20, 712-717) which can give an overview of the total protein expressions of cells are in demand but also overlapping narrow immobilized pH gradients are to be used for more specialized and detailed research and micropreparative separations. The advantage of overlapping narrow pH gradients is the gain in higher resolution by stretching the protein pattern in the first dimension. This simplifies computer-aided image analysis and protein identification (e.g., by mass spectrometry). In this study the protein patterns of yeast cells in pH gradients 4-5, 4.5-5.5, 5-6, 5.5-6.7 and 6-9 are presented and compared to the pH 4-7 and 3-10 gradients. This combination allowed us to reveal a total of 2286 yeast protein spots compared to 755 protein spots in the pH 3-10 gradient.

The recovery of physiological functionality, which is commonly seen in tissue mimetic three-dimensional (3D) cellular aggregates (organoids, spheroids, acini, etc.), has been observed in cells of many origins (primary tissues, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and immortal cell lines). This plurality and plasticity suggest that probably several basic principles promote this recovery process. The aim of this study was to identify these basic principles and describe how they are regulated so that they can be taken in consideration when micro-bioreactors are designed. Here, we provide evidence that one of these basic principles is hypoxia, which is a natural consequence of multicellular structures grown in microgravity cultures. Hypoxia drives a partial metabolic reprogramming to aerobic glycolysis and an increased anabolic synthesis. A second principle is the activation of cytoplasmic glutaminolysis for lipogenesis. Glutaminolysis is activated in the presence of hypo- or normo-glycaemic conditions and in turn is geared to the hexosamine pathway. The reducing power needed is produced in the pentose phosphate pathway, a prime function of glucose metabolism. Cytoskeletal reconstruction, histone modification, and the recovery of the physiological phenotype can all be traced to adaptive changes in the underlying cellular metabolism. These changes are coordinated by mTOR/Akt, p53 and non-canonical Wnt signaling pathways, while myc and NF-kB appear to be relatively inactive. Partial metabolic reprogramming to aerobic glycolysis, originally described by Warburg, is independent of the cell’s rate of proliferation, but is interwoven with the cells abilities to execute advanced functionality needed for replicating the tissues physiological performance.

Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with "standard lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyI)dimethylam~onio]-l -propanesulfonate (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCI, pH 7.0, followed by dilution with "standa r d lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiouredurea lysis buffer (2 M thiourea / 7 M urea, 4% w/v CHAPS, 1% wlv DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i), considerable degradation of high Mr proteins occurred.In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high M, proteins were further improved by pre-boiling with SDS and using thiouredurea lysis buffer instead of "standard lysis buffer (procedure iii).

Natural products, or their synthetic derivatives are a treasure trove to find potential candidates for novel drugs for human treatment. The selection of diamonds from the huge pile of worthless stone is a critical--and difficult--stage in the discovery pipeline. Of all the factors to be considered, perhaps the most important, is that the compound should have the desired effect on the tissue in vivo. Since it is not possible (or ethical) to test all compounds in vivo one must preselect using a surrogate assay system. While animal models have the advantage of being holistic and current 3D culture systems are reductionistic, they at least can be constructed from human cell types. In this review we will consider some of the evidence demonstrating that cells grown in 3D cultures have physiological performances that mimic functions seen in human tissues significantly better than cells grown using classical 2D culture systems. We will discuss advantages and disadvantages of these new culture technologies and highlight theoretical reasons for the differences. 3D cell culture technologies are more labour intensive than 2D culture systems and therefore their introduction is a trade-off between the value of obtaining data that is more relevant to the human condition against their through-put. It is already clear that future in vitro 3D systems will become more complex, using multiple cell types to more faithfully represent a particular tissue or even organ system. And one thing is sure - the diamonds are not easy to find!

The synthesis of early proteins from Chlamydia trachomatis serovar L2 was analyzed by two-dimensional gel electrophoresis. By pulse-label experiments, the synthesis of seven proteins was observed at 2 to 8 h postinfection before the major outer membrane protein was detected at 8 to 10 h after infection. The early proteins were synthesized throughout the 30-h period investigated, but the synthesis of three proteins of 75, 62, and 45 kilodaltons decreased from 26 to 30 h postinfection. Pulse-chase analysis showed that the signals from the same three proteins declined 26 to 30 h after infection. Three of the early proteins were identified as the S1 ribosomal protein, the GroEL-like protein, and DnaK-like protein, respectively.