Calcium block of Na+ channels and its effect on closing rate

Calcium ion transiently blocks Na+ channels, and it shortens the time course for closing of their activation gates. We examined the relation between block and closing kinetics by using the Na+ channels natively expressed in GH3 cells, a clonal line of rat pituitary cells. To simplify analysis, inactivation of the Na+ channels was destroyed by including papain in the internal medium. All divalent cations tested, and trivalent La3+, blocked a progressively larger fraction of the channels as their concentration increased, and they accelerated the closing of the Na+ channel activation gate. For calcium, the most extensively studied cation, there is an approximately linear relation between the fraction of the channels that are calcium-blocked and the closing rate. Extrapolation of the data to very low calcium suggests that closing rate is near zero when there is no block. Analysis shows that, almost with certainty, the channels can close when occupied by calcium. The analysis further suggests that the channels close preferentially or exclusively from the calcium-blocked state.

Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand–DNA complexes: The interaction between DNA and calichearubicin B

We present a helical unwinding assay for reversibly binding DNA ligands that uses closed circular DNA, topoisomerase I (Topo I), and two-dimensional agarose gel electrophoresis. Serially diluted Topo I relaxation reactions at constant DNA/ligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (φ) and intrinsic association constant (Ka). Mathematical treatment of apparent unwinding is formally analogous to that of apparent extinction coefficient data for optical binding titrations. Extrapolation to infinite DNA concentration yields the true unwinding angle of a given ligand and its association constant under Topo I relaxation conditions. Thus this assay delivers simultaneous structural and thermodynamic information describing the ligand–DNA complex. The utility of this assay has been demonstrated by using calichearubicin B (CRB), a synthetic hybrid molecule containing the anthraquinone chromophore of (DA) and the carbohydrate domain of calicheamicin γ1I. The unwinding angle for CRB calculated by this method is −5.3 ± 0.5°. Its Ka value is 0.20 × 106 M−1. For comparison, the unwinding angles of ethidium bromide and DA have been independently calculated, and the results are in agreement with canonical values for these compounds. Although a stronger binder to selected sites, CRB is a less potent unwinder than its parent compound DA. The assay requires only small amounts of ligand and offers an attractive option for analysis of DNA binding by synthetic and natural compounds.

Assigning folds to the proteins encoded by the genome of Mycoplasma genitalium

A crucial step in exploiting the information inherent in genome sequences is to assign to each protein sequence its three-dimensional fold and biological function. Here we describe fold assignment for the proteins encoded by the small genome of Mycoplasma genitalium. The assignment was carried out by our computer server (http://www.doe-mbi.ucla.edu/people/frsvr/frsvr.html), which assigns folds to amino acid sequences by comparing sequence-derived predictions with known structures. Of the total of 468 protein ORFs, 103 (22%) can be assigned a known protein fold with high confidence, as cross-validated with tests on known structures. Of these sequences, 75 (16%) show enough sequence similarity to proteins of known structure that they can also be detected by traditional sequence–sequence comparison methods. That is, the difference of 28 sequences (6%) are assignable by the sequence–structure method of the server but not by current sequence–sequence methods. Of the remaining 78% of sequences in the genome, 18% belong to membrane proteins and the remaining 60% cannot be assigned either because these sequences correspond to no presently known fold or because of insensitivity of the method. At the current rate of determination of new folds by x-ray and NMR methods, extrapolation suggests that folds will be assigned to most soluble proteins in the next decade.

A selective phenomenology of the seismicity of Southern California.

Predictions of earthquakes that are based on observations of precursory seismicity cannot depend on the average properties of the seismicity, such as the Gutenberg-Richter (G-R) distribution. Instead it must depend on the fluctuations in seismicity. We summarize the observational data of the fluctuations of seismicity in space, in time, and in a coupled space-time regime over the past 60 yr in Southern California, to provide a basis for determining whether these fluctuations are correlated with the times and locations of future strong earthquakes in an appropriate time- and space-scale. The simple extrapolation of the G-R distribution must lead to an overestimate of the risk due to large earthquakes. There may be two classes of earthquakes: the small earthquakes that satisfy the G-R law and the larger and large ones. Most observations of fluctuations of seismicity are of the rate of occurrence of smaller earthquakes. Large earthquakes are observed to be preceded by significant quiescence on the faults on which they occur and by an intensification of activity at distance. It is likely that the fluctuations are due to the nature of fractures on individual faults of the network of faults. There are significant inhomogeneities on these faults, which we assume will have an important influence on the nature of self-organization of seismicity. The principal source of the inhomogeneity on the large scale is the influence of geometry--i.e., of the nonplanarity of faults and the system of faults.

The neck region of the myosin motor domain acts as a lever arm to generate movement.

The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain.

Very-long-baseline radio interferometry (VLBI) observations of gamma-ray blazars: results from millimeter-VLBI observations.

VLBI observations of the extremely gamma-bright blazar PKS 0528+134 at 8, 22, 43, and 86 GHz reveal a strongly bent one-sided-core jet structure with at least three moving and two apparently stationary jet components. At the highest observing frequencies the brightest and most compact jet component (the VLBI core) is unresolved with an upper limit to its size of approximately 50 microarcsec corresponding to approximately 0.2 parsec [H0 = 100 km.s-1.Mpc-1 (megaparsec-1), q0 = 0.5, where H0 is Hubble constant and q0 is the deceleration parameter]. Two 86-GHz VLBI observations performed in 1993.3 and 1994.0 reveal a new jet component emerging with superluminal speed from the core. Linear back-extrapolation of its motion yields strong evidence that the ejection of this component is related to an outburst in the millimeter regime and a preceding intense flare of the gamma-flux density observed in early 1993. This and the radio/optical "light curves" and VLBI data for two other sources (S5 0836+710 and 3C 454.3) suggest that the observed gamma-radiation might be Doppler-boosted and perhaps is closely related to the physical processes acting near the "base" of the highly relativistic jets observed in quasars.