Analyzing M-CSF dependent monocyte/macrophage differentiation: Expression modes and meta-modes derived from an independent component analysis

Background  

The analysis of high-throughput gene expression data sets derived from microarray experiments still is a field of extensive investigation. Although new approaches and algorithms are published continuously, mostly conventional methods like hierarchical clustering algorithms or variance analysis tools are used. Here we take a closer look at independent component analysis (ICA) which is already discussed widely as a new analysis approach. However, deep exploration of its applicability and relevance to concrete biological problems is still missing. In this study, we investigate the relevance of ICA in gaining new insights into well characterized regulatory mechanisms of M-CSF dependent macrophage differentiation.     

Topological analysis of plasmid chromatin from yeast and mammalian cells

Yeast has proven to be a powerful system for investigation of chromatin structure. However, the extent to which yeast chromatin can serve as a model for mammalian chromatin is limited by the significant number of differences that have been reported. To further investigate the structural relationship between the two chromatins, we have performed a DNA topological analysis of pRSSVO, a 5889 base-pair plasmid that can replicate in either yeast or mammalian cells. When grown in mammalian cells, pRSSVO contains an average of 33 negative supercoils, consistent with one nucleosome per 181 bp. This is close to the measured nucleosome repeat length of 190 bp. However, when grown in yeast cells, pRSSVO contains an average of only 23 negative supercoils, which is indicative of only one nucleosome per 256 bp. This is dramatically different from the measured nucleosome repeat length of 165 bp. To account for these observations, we suggest that yeast chromatin is composed of relatively short ordered arrays of nucleosomes with a repeat of 165 bp, separated by substantial gaps, possibly corresponding to regulatory regions.     

Constitutive Nuclear Localization of an Alternatively Spliced Sirtuin-2 Isoform

Sirtuin-2 (SIRT2), the cytoplasmic member of the sirtuin family, has been implicated in the deacetylation of nuclear proteins. Although the enzyme has been reported to be located to the nucleus during G₂/M phase, its spectrum of targets suggests functions in the nucleus throughout the cell cycle. While a nucleocytoplasmic shuttling mechanism has been proposed for SIRT2, recent studies have indicated the presence of a constitutively nuclear isoform. Here we report the identification of a novel splice variant (isoform 5) of SIRT2 that lacks a nuclear export signal and encodes a predominantly nuclear isoform. This novel isoform 5 fails to show deacetylase activity using several assays, both in vitro and in vivo, and we are led to conclude that this isoform is catalytically inactive. Nevertheless, it retains the ability to interact with p300, a known interaction partner. Moreover, changes in intrinsic tryptophan fluorescence upon denaturation indicate that the protein is properly folded. These data, together with computational analyses, confirm the structural integrity of the catalytic domain. Our results suggest an activity-independent nuclear function of the novel isoform.     

Methodology and Indicators of Economy‐wide Material Flow Accounting

This contribution presents the state of the art of economy‐wide material flow accounting. Starting from a brief recollection of the intellectual and policy history of this approach, we outline system definition, key methodological assumptions, and derived indicators. The next section makes an effort to establish data reliability and uncertainty for a number of existing multinational (European and global) material flow accounting (MFA) data compilations and discusses sources of inconsistencies and variations for some indicators and trends. The results show that the methodology has reached a certain maturity: Coefficients of variation between databases lie in the range of 10% to 20%, and correlations between databases across countries amount to an average R² of 0.95. After discussing some of the research frontiers for further methodological development, we conclude that the material flow accounting framework and the data generated have reached a maturity that warrants material flow indicators to complement traditional economic and demographic information in providing a sound basis for discussing national and international policies for sustainable resource use.     

C3G/Rapgef1 Is Required in Multipolar Neurons for the Transition to a Bipolar Morphology during Cortical Development

The establishment of a polarized morphology is essential for the development and function of neurons. During the development of the mammalian neocortex, neurons arise in the ventricular zone (VZ) from radial glia cells (RGCs) and leave the VZ to generate the cortical plate (CP). During their migration, newborn neurons first assume a multipolar morphology in the subventricular zone (SVZ) and lower intermediate zone (IZ). Subsequently, they undergo a multi-to-bipolar (MTB) transition to become bipolar in the upper IZ by developing a leading process and a trailing axon. The small GTPases Rap1A and Rap1B act as master regulators of neural cell polarity in the developing mouse neocortex. They are required for maintaining the polarity of RGCs and directing the MTB transition of multipolar neurons. Here we show that the Rap1 guanine nucleotide exchange factor (GEF) C3G (encoded by the Rapgef1 gene) is a crucial regulator of the MTB transition in vivo by conditionally inactivating the Rapgef1 gene in the developing mouse cortex at different time points during neuronal development. Inactivation of C3G results in defects in neuronal migration, axon formation and cortical lamination. Live cell imaging shows that C3G is required in cortical neurons for both the specification of an axon and the initiation of radial migration by forming a leading process.     

Purification and characterization of 50S ribosomal proteins of Escherichia coli

Summary Twenty-seven proteins of the 50S ribosomal subunit from E. coli have been purified by a combination of differential solubility in ammonium sulfate, ion-exchange chromatography, and molecular-sieve chromatography. The amino acid compositions, tryptic peptides and molecular weights of these proteins have been analyzed. Each protein is unique with respect to amino acid sequence and, according to chemical criteria, reasonably pure. The sum of the molecular weights of the twenty-seven proteins is 495000. This means that the 50S subunit could accommodate one copy of each protein.     

Affinity isolation of RNA polymerase II on amanitin-Sepharose

We report here the first case of an affinity isolation of eukaryotic RNA polymerase II. The procedure employs an affinity matrix composed of α-amanitin coupled to Sepharose 4B via a ten atom spacer. RNA polymerase II from either calf thymus or wheat germ binds to the amanitin-Sepharose, as indicated by subsequent elution with sodium dodecylsulfate-containing buffer and analysis by polyacrylamide gel electrophoresis. The specificity of binding is demonstrated by the fact that when the enzyme is preincubated with 1 μg/ml of free α-amanitin, subsequent binding to the amanitin-Sepharose is abolished. Elution methods that should permit the recovery of active enzyme from the column are discussed.