Gene mapping on mouse chromosome 8 by interspecific crosses: new data on a linkage group conserved on human chromosome 16q

A large conserved linkage group exists on mouse chromosome 8 and human chromosome 16q, including the loci for chymotrypsinogen B (Ctrb), haptoglobin (Hp), lecithin:cholesterol acyltransferase (Lcat), metallothionein-1,-2 (Mt-1,-2), tyrosine aminotransferase (Tat), and uvomorulin (Um). Using cloned gene probes, these six loci were mapped in M. m. domesticus X M. spretus interspecific crosses relative to a number of chromosome 8 anchor loci resulting in the gene order Es-1,Es-9-Mt-1,-2-Got-2-Es-2,Es-7,Lcat,Um-Hp,Tat,Ctrb-e. These results complement earlier studies and redefine the conserved segment on mouse chromosome 8, previously defined by the Hp-Tat interval, by the 24-cM interval between Mt-1,-2 and the conserved locus for adenine phosphoribosyltransferase, Aprt, mapped at 25 cM from Es-1 by T. B. Nesterova, P. M. Borodin, S. M. Zakian, and O. L. Serov (1987, Biochem. Genet. 25: 563-568). Within this segment, the gene order appears the same in man and mouse. While map distances between HP-TAT,HP-CTRB, and TAT-CTRB of respectively 7, 11, and 9 cM have previously been measured in man, no crossovers between Hp, Tat, and Ctrb were observed in over 100 meioses in the mouse.     

A Novel Microtubule Inhibitor 4SC-207 with Anti-Proliferative Activity in Taxane-Resistant Cells

Microtubule inhibitors are invaluable tools in cancer chemotherapy: taxanes and vinca alkaloids have been successfully used in the clinic over the past thirty years against a broad range of tumors. However, two factors have limited the effectiveness of microtubule inhibitors: toxicity and resistance. In particular, the latter is highly unpredictable, variable from patient to patient and is believed to be the cause of treatment failure in most cases of metastatic cancers. For these reasons, there is an increasing demand for new microtubule inhibitors that can overcome resistance mechanisms and that, at the same time, have reduced side effects. Here we present a novel microtubule inhibitor, 4SC-207, which shows strong anti-proliferative activity in a large panel of tumor cell lines with an average GI₅₀ of 11nM. In particular, 4SC-207 is active in multi-drug resistant cell lines, such as HCT-15 and ACHN, suggesting that it is a poor substrate for drug efflux pumps. 4SC-207 inhibits microtubule growth in vitro and in vivo and promotes, in a dose dependent manner, a mitotic delay/arrest, followed by apoptosis or aberrant divisions due to chromosome alignment defects and formation of multi-polar spindles. Furthermore, preliminary data from preclinical studies suggest low propensity towards bone marrow toxicities at concentrations that inhibit tumor growth in paclitaxel-resistant xenograft models. In summary, our results suggest that 4SC-207 may be a potential anti-cancer agent.     

The Small Heat Shock Protein Hsp27 Affects Assembly Dynamics and?Structure of Keratin Intermediate Filament Networks

The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation.     

Conserved intron positions in ancient protein modules

Background  

The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt) derived from GenBank.     

Interactions of the N-terminal domain of ribosomal protein L11 with thiostrepton and rRNA

Ribosomal protein L11 has two domains: the C-terminal domain (L11-C76) binds rRNA, whereas the N-terminal domain (L11-NTD) may variously interact with elongation factor G, the antibiotic thiostrepton, and rRNA. To begin to quantitate these interactions, L11 from Bacillus stearothermophilus has been overexpressed and its properties compared with those of L11-C76 alone in a fluorescence assay for protein-rRNA binding. The assay relies on 2'-amino-butyryl-pyrene-uridine incorporated in a 58-nucleotide rRNA fragment, which gives approximately 15-fold enhancement when L11 or L11-C76 is bound. Although the pyrene tag weakens protein binding, unbiased protein-RNA association constants were obtained in competition experiments with untagged RNA. It was found that (i) intact B. stearothermophilus L11 binds rRNA with K approximately 1.2 x 10(9) m(-1) in buffers with 0.2 m KCl, about 100-fold tighter than Escherichia coli L11; (ii) the N-terminal domain makes a small, salt-dependent contribution to the overall L11-RNA binding affinity (approximately 8-fold enhancement at 0.2 m KCl), (iii) L11 stimulates thiostrepton binding by 2.3 +/- 0.6 x 10(3)-fold, predicting an overall thiostrepton affinity for the ribosome of approximately 10(9) m(-1), and (iv) the yeast homolog of L11 shows no stimulation of thiostrepton binding. The latter observation resolves the question of why eukaryotes are insensitive to the antibiotic. These measurements also show that it is plausible for thiostrepton to compete directly with EF-G.GDP for binding to the L11-RNA complex, and provide a quantitative basis for further studies of L11 function and thiostrepton mechanism.     

Sepsis progression and outcome: a dynamical model

Background  

Sepsis (bloodstream infection) is the leading cause of death in non-surgical intensive care units. It is diagnosed in 750,000 US patients per annum, and has high mortality. Current understanding of sepsis is predominately observational and correlational, with only a partial and incomplete understanding of the physiological dynamics underlying the syndrome. There exists a need for dynamical models of sepsis progression, based upon basic physiologic principles, which could eventually guide hourly treatment decisions.     

Contraction mechanisms in composite active actin networks

Simplified in vitro systems are ideally suited for studying the principle mechanisms of the contraction of cytoskeletal actin systems. To shed light on the dependence of the contraction mechanism on the nature of the crosslinking proteins, we study reconstituted in vitro active actin networks on different length scales ranging from the molecular organization to the macroscopic contraction. Distinct contraction mechanisms are observed in polar and apolar crosslinked active gels whereas composite active gels crosslinked in a polar and apolar fashion at the same time exhibit both mechanisms simultaneously. In polar active actin/fascin networks initially bundles are formed which are then rearranged. In contrast, apolar cortexillin-I crosslinked active gels are bundled only after reorganization of actin filaments by myosin-II motor filaments.     

Assessing sub-seafloor microbial activity by combined stable isotope probing with deuterated water and 13C-bicarbonate

Sub‐seafloor sediments are populated by large numbers of microbial cells but not much is known about their metabolic activities, growth rates and carbon assimilation pathways. Here we introduce a new method enabling the sensitive detection of microbial lipid production and the distinction of auto‐ and heterotrophic carbon assimilation. Application of this approach to anoxic sediments from a Swedish fjord allowed to compare the activity of different functional groups, the growth and turnover times of the bacterial and archaeal communities. The assay involves dual stable isotope probing (SIP) with deuterated water (D₂O) and ¹³C_DIC (d issolved i norganic c arbon). Culture experiments confirmed that the D content in newly synthesized lipids is in equilibrium with the D content in labelled water, independent of whether the culture grew hetero‐ or autotrophically. The ratio of ¹³C_DIC to D₂O incorporation enables distinction between these two carbon pathways in studies of microbial cultures and in environmental communities. Furthermore, D₂O‐SIP is sufficiently sensitive to detect the formation of few hundred cells per day in a gram of sediment. In anoxic sediments from a Swedish fjord, we found that > 99% of newly formed lipids were attributed to predominantly heterotrophic bacteria. The production rate of bacterial lipids was highest in the top 5 cm and decreased 60‐fold below this depth while the production rate of archaeal lipids was rather low throughout the top meter of seabed. The contrasting patterns in the rates of archaeal and bacterial lipid formation indicate that the factors controlling the presence of these two lipid groups must differ fundamentally.     

Cytoskeletal bundle mechanics

The mechanical properties of cytoskeletal actin bundles play an essential role in numerous physiological processes, including hearing, fertilization, cell migration, and growth. Cells employ a multitude of actin-binding proteins to actively regulate bundle dimensions and cross-linking properties to suit biological function. The mechanical properties of actin bundles vary by orders of magnitude depending on diameter and length, cross-linking protein type and concentration, and constituent filament properties. Despite their importance to cell function, the molecular design principles responsible for this mechanical behavior remain unknown. Here, we examine the mechanics of cytoskeletal bundles using a molecular-based model that accounts for the discrete nature of constituent actin filaments and their distinct cross-linking proteins. A generic competition between filament stretching and cross-link shearing determines three markedly different regimes of mechanical response that are delineated by the relative values of two simple design parameters, revealing the universal nature of bundle-bending mechanics. In each regime, bundle-bending stiffness displays distinct scaling behavior with respect to bundle dimensions and molecular composition, as observed in reconstituted actin bundles in vitro. This mechanical behavior has direct implications on the physiological bending, buckling, and entropic stretching behavior of cytoskeletal processes, as well as reconstituted actin systems. Results are used to predict the bending regimes of various in vivo cytoskeletal bundles that are not easily accessible to experiment and to generate hypotheses regarding implications of the isolated behavior on in vivo bundle function.