Caldesmon Inhibits Nonmuscle Cell Contractility and Interferes with the Formation of Focal Adhesions

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca2+–calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca2+–calmodulin or Ca2+–S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

Morphogenetic Effects of Neuregulin (Neu Differentiation Factor) in Cultured Epithelial Cells

Neuregulin, or neu differentiation factor, induces cell proliferation or differentiation through interaction with members of the ErbB family of receptor tyrosine kinases. We report that neuregulin can also induce profound morphogenic responses in cultured epithelial cells of different origins. These effects include scattering of small epithelial islands and rearrangement of larger cell islands into ordered ring-shaped arrays with internal lumens. The ring-forming cells are interconnected by cadherin- and β-catenin-containing adherens junctions. In confluent cultures, neuregulin treatment induces formation of circular lumenlike gaps in the monolayer. Both cell scattering and ring formation are accompanied by a marked increase in cell motility that is independent of hepatocyte growth factor/scatter factor and its receptor (c-Met). Affinity-labeling experiments implied that a combination of ErbB-2 with ErbB-3 mediates the morphogenic signal of neuregulin in gastric cells. Indeed, a similar morphogenic effect could be reconstituted in nonresponsive cells by coexpression of ErbB-2 and -3. We conclude that a heterodimer between the kinase-defective neuregulin receptor, ErbB-3, and the coreceptor, ErbB-2, mediates the morphogenetic action of neuregulin.

In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries.

A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (< or = 0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.

Increased fibrinogen levels at diagnosis are associated with adverse outcome in patients with acute myeloid leukemia

Increased plasma fibrinogen levels are associated with shortened overall survival (OS) in some solid tumor types. In contrast, the prognostic significance of varying fibrinogen levels in acute myeloid leukemia (AML) at diagnosis is unknown. In this study, we assessed the prognostic significance of fibrinogen levels in AML patients. In a comprehensive retrospective single-center study, we determined the survival rates of 375 consecutive AML patients undergoing at least one cycle of intensive chemotherapy induction treatment. Patients were dichotomized between low (<4.1 g/L) and high fibrinogen levels (≥4.1 g/L) at diagnosis of AML before initiation of treatment. Subsequently, quartile ranges were applied to analyze the association of varying fibrinogen levels on survival. We observed that the rates of complete remission, early death, and admission to intensive care unit were equal in the low versus high fibrinogen group. However, OS was significantly better in the low fibrinogen group (27.3 vs 13.5 months; p = 0.0009) as well as progression-free survival (12.3 vs 7.8 months; p = 0.0076). This survival difference remained significant in the multivariate analysis (p = 0.003). Assessing quartiles of fibrinogen values, we further confirmed this observation. Our data suggest that high fibrinogen levels at diagnosis of AML are associated with unfavorable OS and progression-free survival but not with increased mortality during induction treatment.

The Topolovampo Pacific railway : a commercial and statistical review, showing its local, transcontinental, international and interoceanic features and advantages /

Bound in a vol. with supplied title: [Austin-Topolovampo railroad documents]