Integrating the actin and vimentin cytoskeletons. adhesion-dependent formation of fimbrin-vimentin complexes in macrophages.

Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH(2)-terminal domain of vimentin and the vimentin binding site at residues 143-188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin-vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.     

Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C⁺ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pK_a of an iM. We established that the C-C⁺ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.     

Studies on biomarkers of copper exposure and toxicity in the marine amphipod Gammarus locusta (Crustacea): I. Induction of metallothionein and lipid peroxidation

Sublethal exposures of the marine amphipod Gammarus locusta to a concentration range of copper (Cu) in water (4 days' exposure; 3, 5 and 10 μg Cu l^-1) or spiked sediments (28 days' exposure; 1, 3 and 6 mg Cu kg^-1 dry weight) were performed, and the resulting bioaccumulation of Cu and effects on putative metallothionein (MT) and lipid peroxidation (LP) were investigated. A time-course exposure study (over 10 days) to a single water-borne concentration of Cu (4 μg l^-1) was also carried out. MT and LP were quantified, respectively, by differential pulse polarography and as thiobarbituric acid-reactive malondialdehyde equivalents. The increasing levels of Cu in water and sediment exposures resulted in enhanced uptake of the metal by G. locusta. Synthesis of putative MT occurred in response to exposure to water-borne Cu, the levels being higher (p < 0.05) over the dose range of Cu compared with controls. A positive correlation was observed between putative MT levels and the Cu body-burden concentration (p < 0.001). However, no increase in LP was observed in these animals. In contrast, in the time-course experiment, LP levels increased within 1 day of exposure, subsequently peaking at 4 days (68% greater than control, p < 0.001), before returning to control values by day 6. Higher levels of MT were also observed in this exposure, but at days 6 and 10 (55% and 38%, respectively), paralleling the decrease in LP. No increase in MT levels was recorded with exposure to Cu-contaminated sediments, whereas higher levels of LP were seen in comparison with controls (p < 0.001). Overall, the inverse relationship between putative MT induction and the occurrence of LP indicates that MT may protect against the prooxidant effects of Cu. It is concluded that MT and LP offer potential for application as biomarkers in G. locusta.     

Role of Sonic hedgehog signaling in cell cycle,oxidative stress,and autophagy of temozolomide resistant glioblastoma

The first-line chemotherapy treatment for Glioblastoma (GBM) - the most aggressive and frequent brain tumor - is temozolomide (TMZ). The Sonic hedgehog (SHH) pathway is involved with GBM tumorigenesis and TMZ chemoresistance. The role of SHH pathway inhibition in the potentiation of TMZ's effects using T98G, U251, and GBM11 cell lines is investigated herein. The combination of GANT-61 and TMZ over 72 hr suggested a synergistic effect. All TMZ-resistant cell lines displayed a significant decrease in cell viability, increased DNA fragmentation and loss of membrane integrity. For T98G cells, G₂/M arrest was observed, while U251 cells presented a significant increase in reactive oxygen species production and catalase activity. All the cell lines presented acidic vesicles formation correlated to Beclin-1 overexpression. The combined treatment also enhanced GLI1 expression, indicating the presence of select resistant cells. The selective inhibition of the SHH pathway potentiated the cytotoxic effect of TMZ, thus becoming a promising in vitro strategy for GBM treatment.     

Length–weight relationships of five selected demersal fishes from the Cabo Verde Islands (eastern-central Atlantic)

The length–weight relationships (LWR) were estimated for five selected demersal marine fishes from the relatively undisturbed shelves and slopes of the Cabo Verde Islands. Most of them were of ecological and/or commercial interest. Occasional sampling was carried out during different fisheries research projects. LWRs were taken for the following bycatch species for which no such estimates exists in the science literature: Myroconger compressus, Synaphobranchus affinis, Physiculus cyanostrophus, Physiculus caboverdensis and Canthidermis maculata. Total length and fork length (to the nearest 0.1 cm) and total weight (to the nearest 0.1 g) were determined. The resulting length–weight equations are characterized by a high accompanying coefficient of correlation. The present data help improving the knowledge base needed for further fish biology studies and fisheries management.