Calcium-mediated inactivation of the calcium conductance in cesium- loaded frog heart cells

Ca current inactivation was investigated in frog atrial muscle under voltage-clamp conditions. To inhibit the outward currents, experiments were performed on Cs-loaded fibers and in 20 mM Cs (K-free) Ringer with 4-AP added. Inactivation, produced by a conditioning pulse, was measured by reducing the current during a subsequent test pulse. The extent of inactivation increased initially with prepulse amplitude and then decreased as the prepulse potential became progressively positive. Relative inactivation follows a U-shaped curve. When Sr was substituted for Ca, both the degree and the rate of inactivation decreased. Relative inactivation appeared to be linearly related to the amount of divalent cations (Ca and Sr) carried into the cell during the prepulse. Elevating Ca enhanced peak current and accelerated its decline. Elevating Mg decreased peak current and slowed its decline. An application of Na-free (LiCl) solution resulted in a somewhat smaller but faster inactivating current. Adrenaline increased and D600 decreased the maximal Ca conductance with little alteration in the inactivation rate; Co decreased both peak current and the rate of inactivation. Enhancement of the outward currents, reduced driving force, and intracellular surface charge screening do not adequately account for the above results. Evidence was considered that Ca entry mediates most of Ca current inactivation in frog atrial fibers. Removal from inactivation was also investigated in normal-Ca, Ca-rich, and Sr solutions. Recovery after partial inactivation by high depolarization was biphasic. Recovery was slowed by 10 Ca and accelerated by 1.8 Sr, whereas opposite effects have been shown on activation.     

An estimation of the genome length of maritime pine (Pinus pinaster Ait.)

The genome length, in units of Morgans or centimorgans, is a fundamental feature of a species. It can be calculated from a complete linkage map. However, the genome size can be estimated with partial linkage data. Using linkage data obtained by the analysis of a two-dimensional electrophoresis of the proteins contained in an haploid organ, the megagametophyte, we suggest an estimation and a confidence interval of the genome length of a gymnosperm, the maritime pine (Pinus pinaster Ait.). The results indicate an important gap between the physical and the genetic maps.     

Slow inward Ca current in frog heart: theoretical evidence against a voltage-dependent inactivation

The validity of a Hodgkin-Huxley type voltage-dependent inactivation of slow inward Ca current (Isi) was tested in frog heart using a computer simulation. The time course of Isi was calculated during the development of a frog atrial action potential (AP). With a time constant of inactivation (tauf) of 55 ms at a membrane potential (Em) of -15 mV, the variation of Isi was biphasic: after a transient increase followed by a decrease to zero, Isi partially "reactivated" (at the beginning of the AP repolarization phase) and then fully deactivated. The "reactivation" phase of Isi developed whether tauf was an increasing, decreasing, U-shaped, or bell-shaped function of Em. The addition of an independent and slower process responsible for the recovery from inactivation only partly suppressed the "reactivation" phase. However, until now there was no experimental evidence supporting such a biphasic variation of Isi during AP repolarization. Thus our results indicate that the Hodgkin-Huxley type model of the voltage-dependence of Isi-inactivation process may not correctly represent the actual behavior of frog cardiac muscle.     

Inhibition by glucagon of the cGMP-inhibited low-Km cAMP phosphodiesterase in heart is mediated by a pertussis toxin-sensitive G-protein.

We have recently reported that glucagon activated the L-type Ca2+ channel current in frog ventricular myocytes and showed that this was linked to the inhibition of a membrane-bound low-Km cAMP phosphodiesterase (PDE) (Méry, P. F., Brechler, V., Pavoine, C., Pecker, F., and Fischmeister, R. (1990) Nature 345, 158-161). We show here that the inhibition of membrane-bound PDE activity by glucagon depends on guanine nucleotides, a reproducible inhibition of 40% being obtained with 0.1 microM glucagon in the presence of 10 microM GTP, with GTP greater than GTP gamma S, while GDP and ATP gamma S were without effect. Glucagon had no effect on the cytosolic low-Km cAMP PDE, assayed with or without 10 microM GTP. Glucagon inhibition of membrane-bound PDE activity was not affected by pretreatment of the ventricle particulate fraction with cholera toxin. However, it was abolished after pertussis toxin pretreatment. Mastoparan, a wasp venom peptide known to activate G(i)/G(o) proteins directly, mimicked the effect of glucagon. PDE inhibition by glucagon was additive with the inhibition induced by Ro 20-1724, but was prevented by milrinone. This was correlated with an increase by glucagon of cAMP levels in frog ventricular cells which was not additive with the increase in cAMP due to milrinone. We conclude that glucagon specifically inhibits the cGMP-inhibited, milrinone-sensitive PDE (CGI-PDE). Insensitivity of adenylylcyclase to glucagon and inhibition by the peptide of a low-Km cAMP PDE were not restricted to frog heart, but also occurred in mouse and guinea pig heart. These results confirm that two mechanisms mediate the action of glucagon in heart: one is the activation of adenylylcyclase through Gs, and the other relies on the inhibition of the membrane-bound low-Km CGI-PDE, via a pertussis toxin-sensitive G-protein.     

Native drivers of fish life history traits are lost during the invasion process

Rapid adaptation to global change can counter vulnerability of species to population declines and extinction. Theoretically, under such circumstances both genetic variation and phenotypic plasticity can maintain population fitness, but empirical support for this is currently limited. Here, we aim to characterize the role of environmental and genetic diversity, and their prior evolutionary history (via haplogroup profiles) in shaping patterns of life history traits during biological invasion. Data were derived from both genetic and life history traits including a morphological analysis of 29 native and invasive populations of topmouth gudgeon Pseudorasbora parva coupled with climatic variables from each location. General additive models were constructed to explain distribution of somatic growth rate (SGR) data across native and invasive ranges, with model selection performed using Akaike's information criteria. Genetic and environmental drivers that structured the life history of populations in their native range were less influential in their invasive populations. For some vertebrates at least, fitness‐related trait shifts do not seem to be dependent on the level of genetic diversity or haplogroup makeup of the initial introduced propagule, nor of the availability of local environmental conditions being similar to those experienced in their native range. As long as local conditions are not beyond the species physiological threshold, its local establishment and invasive potential are likely to be determined by local drivers, such as density‐dependent effects linked to resource availability or to local biotic resistance.     

In vitro reconstitution of Cascade‐mediated CRISPR immunity in Streptococcus thermophilus

Clustered regularly interspaced short palindromic repeats (CRISPR)‐encoded immunity in Type I systems relies on the Cascade (CRISPR‐associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4‐Cas (CRISPR‐associated) system (St‐CRISPR4‐Cas), we isolated an effector complex (St‐Cascade) containing 61‐nucleotide CRISPR RNA (crRNA). We show that St‐Cascade, guided by crRNA, binds in vitro to a matching proto‐spacer if a proto‐spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (?1 position) of the proto‐spacer. In the presence of a correct PAM, St‐Cascade binding to the target DNA generates an R‐loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R‐loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St‐CRISPR4‐Cas and other Type I systems.