Distance measurements in cardiac troponin C

Intramolecular distance measurements were made in cardiac troponin C (cTnC) by fluorescence energy transfer using Eu3+ or Tb3+ as energy donors and Nd3+ or an organic chromophore as acceptors. The laser-induced luminescence of bound Eu3+ is quenched in Eu1Nd1cTnC with a lifetime of 0.328 ms, compared with 0.43 ms for Eu2cTnC. The enhanced decay corresponds to an energy transfer efficiency of 0.25, or a distance of 1.1 nm between the two high affinity sites. We have also labeled cTnC with 4-dimethylaminophenylazophenyl-4'-maleimide (DAB-Mal) at the two cysteine residues (Cys-35 and Cys-84). Energy transfer measurements were carried out between Tb3+ bound to the high affinity sites and the labels attached to the domain containing the low affinity site. Upon uv irradiation at pH 6.7, Tb1cTnCDAB emits tyrosine-sensitized Tb3+ luminescence that decays bioexponentially with lifetimes of 1.29 and 0.76 ms. The shorter lifetime is ascribed to energy transfer from Tb3+ to the DAB labels, yielding an average distance of 3.4 nm between the donor and the acceptors. At pH 5.0, however, the luminescence decays exclusively with a single lifetime of 1.31 ms, suggesting that under these conditions all Tb3+ ions are more than 5.2 nm away from the label. Thus cTnC, like skeletal TnC, undergoes a pH-dependent conformational transition which converts an elongated structure at lower pH's to a rather compact conformation in a more physiological medium.     

Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent

Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.     

17beta-Estradiol responsiveness of MCF-7 laboratory strains is dependent on an autocrine signal activating the IGF type I receptor

BACKGROUND: Human MCF-7 cells have been studied extensively as a model for breast cancer cell growth. Many reports have established that serum-starved MCF-7 cells can be induced to proliferate upon the sole addition of 17beta-estradiol (E2). However, the extent of the mitogenic response to E2 varies in different MCF-7 strains and may even be absent. In this study we compared the E2-sensitivity of three MCF-7 laboratory strains. RESULTS: The MCF-7S line is non-responsive to E2, the MCF-7 ATCC has an intermediate response to E2, while the MCF-7 NKI is highly E2-sensitive, although the levels and activities of the estrogen receptor (ER) are not significantly different. Both suramin and IGF type I receptor blocking antibodies are able to inhibit the mitogenic response to E2-treatment in MCF-7 ATCC and MCF-7 NKI cells. From this we conclude that E2-induced proliferation is dependent on IGF type I receptor activation in all three MCF-7 strains. CONCLUSIONS: The results presented in this article suggest that E2-responsiveness of MCF-7 cells is dependent on the secretion of an autocrine factor activating the IGF-IR. All three strains of MCF-7 breast cancer cells investigated do not respond to E2 if the IGF-RI-pathway is blocked. Generally, breast cancer therapy is targeted at inhibiting estrogen action. This study suggests that inhibition of IGF-action in combination with anti-estrogen-treatment may provide a more effective way in treatment or even prevention of breast cancer.     

Redescription and palaeobiology of Palaeoscorpius devonicus Lehmann, 1944 from the Lower Devonian Hunsrück Slate of Germany

Abstract: Palaeoscorpius devonicus Lehmann, 1944 is known from only a single specimen, found in the Eschenbach Pit near Bundenbach in the Lower Devonian Hunsrück Slate of Germany. It is a key fossil, having been interpreted both as the most basal member of the Scorpiones and as one of the order’s most likely candidates for an aquatic mode of life. Prepared both ventrally and dorsally, some aspects of its morphology remain problematic. Here, with the aid of new techniques, including computed tomography, we present a re‐investigation of this scorpion’s anatomy and a new reconstruction, with a particular focus on the species’ original habitat. On the basis of the environmental interpretation of the Hunsrück Slate and the completeness of the specimen, previous authors concluded that P. devonicus was marine, but none offered convincing morphological evidence. Recent studies of the deposit’s environment suggest that the Hunsrück Sea was part of an intrashelf basin, relatively close to the coastline, and fossils of land plants show that terrestrial wash‐in occasionally occurred. Our revised interpretation of the fossil’s morphology demonstrates that the scorpion was most probably terrestrial. Internal mesosomal organs are interpreted as book lungs, but other terrestrial adaptations are lacking. The absence of both coxapophyses and gnathobases makes determining the scorpion’s feeding mechanism difficult. Interpreting the scorpion’s character states within a phylogenetic framework, especially the possible presence of book lungs, implies either that the plesiomorphic position of P. devonicus is no longer supported or that the development of book lungs had already taken place early in the scorpion lineage.     

Demographics and landscape features determine intrariver population structure in Atlantic salmon (Salmo salar L.): the case of the River Moy in Ireland

Contemporary genetic structure of Atlantic salmon (Salmo salar L.) in the River Moy in Ireland is shown here to be strongly related to landscape features and population demographics, with populations being defined largely by their degree of physical isolation and their size. Samples of juvenile salmon were collected from the 17 major spawning areas on the river Moy and from one spawning area in each of five smaller nearby rivers. No temporal allele frequency differences were observed within locations for 12 microsatellite loci, whereas nearly all spatial samples differed significantly, suggesting that each was a separate population. Bayesian clustering and landscape genetic analyses suggest that these populations can be combined hierarchically into five genetically informative larger groupings. Lakes were found to be the single most important determinant of the observed population structure. Spawning area size was also an important factor. The salmon population of the closest nearby river resembled genetically the largest Moy population grouping. In addition, we showed that anthropogenic influences on spawning habitats, in this case arterial drainage, can affect relationships between populations. Our results show that Atlantic salmon biodiversity can be largely defined by geography, and thus, knowledge of landscape features (for example, as characterized within Geographical Information Systems) has the potential to predict population structure in other rivers without an intensive genetic survey, or at least to help direct sampling. This approach of combining genetics and geography, for sampling and in subsequent statistical analyses, has wider application to the investigation of population structure in other freshwater/anadromous fish species and possibly in marine fish and other organisms.     

Variants of Myxococcus virescens Exhibiting Dispersed Growth. Growth and Production of Extracellular Enzymes and Slime

The growth of two strains of Myxococcus virescens exhibiting dispersed growth was followed in casamino acids (N III-C) media and casitone media. The changes in optical density, pH, pigmentation as well as the secretion of bacteriolytic and proteolytic enzymes, DNA and polysaccharides during growth were recorded. In both media the bacteria grew exponentially with a generation time of 4 (casitone) and 20 hours (N III-C) respectively. The maximal cell mass was about 4 times higher in casitone than in casamino acids media. The amounts of bacteriolytic enzymes produced by the two strains in N III-C medium were different but in casitone medium they were about equal and considerably higher. The maximal values of proteolytic enzymes were about the same in both media and always occurred later than the bacteriolytic maxima. Both activity peaks appeared before the phase of decline. The polysaccharide production reached a maximum during the stationary growth phase in both media. A higher value was reached during growth in casitone medium than in N III-C medium. During the phase of decline a second increase of polysaccharide in the medium appeared. No DNA could be detected in the cell-free solutions until the beginning of the phase of decline.     

Proton magnetic resonance studies on peptide fragments of troponin-C containing single calcium-binding sites

Proton magnetic resonance spectroscopy has been employed to study the solution conformation of three cleavage fragments of troponin-C, each containing a single Ca(II)-binding site and corresponding to different regions in the primary sequence; viz. CB8 (residues 46–77), CB9 (residues 85–134) and TH2 (residues 121–159). Although all three peptides lack a well-defined tertiary fold in the absence of metal ions, several spectral features indicate the presence of local conformational constraints in each apopeptide. Ca(II) binding led to spectral changes consistent with increased restriction of backbone motility and the adoption of a more compact conformation. Studies using paramagnetic ions as conformational probes support current views concerning the nature of the ligands at the metal binding sites.The nature and kinetics of the structural influence of metal binding suggest that the conformational constraints existing in the CB8 apo-peptide provide an adequate Ca(II)-binding configuration. In contrast, the CB9 and TH2 peptides exhibit spectral changes consistent with an increased local structure in the region of helix E (residues 94–102) in the case of CB9 and helix H (residues 148–159) in the case of TH2. In CB9, conformation changes also appear to be transmitted to a portion of the sequence (residues 87–93) preceding helix E, a putative site of interaction between troponin-C and troponin-I. These data are discussed with reference to the contribution of long-range (interdomain) interactions within troponin-C and the modulation of troponin subunit protein-protein interactions by Ca(II) binding.