Thermodynamic and kinetic properties of amino-terminal and S4-S5 loop HERG channel mutants under steady-state conditions

Gating kinetics and underlying thermodynamic properties of human ether-a-go-go-related gene (HERG) K⁺ channels expressed in Xenopus oocytes were studied using protocols able to yield true steady-state kinetic parameters. Channel mutants lacking the initial 16 residues of the amino terminus before the conserved eag/PAS region showed significant positive shifts in activation voltage dependence associated with a reduction of z_g values and a less negative ΔG_o, indicating a deletion-induced displacement of the equilibrium toward the closed state. Conversely, a negative shift and an increased ΔG_o, indicative of closed-state destabilization, were observed in channels lacking the amino-terminal proximal domain. Furthermore, accelerated activation and deactivation kinetics were observed in these constructs when differences in driving force were considered, suggesting that the presence of distal and proximal amino-terminal segments contributes in wild-type channels to specific chemical interactions that raise the energy barrier for activation. Steady-state characteristics of some single point mutants in the intracellular loop linking S4 and S5 helices revealed a striking parallelism between the effects of these mutations and those of the amino-terminal modifications. Our data indicate that in addition to the recognized influence of the initial amino-terminus region on HERG deactivation, this cytoplasmic region also affects activation behavior. The data also suggest that not only a slow movement of the voltage sensor itself but also delaying its functional coupling to the activation gate by some cytoplasmic structures possibly acting on the S4-S5 loop may contribute to the atypically slow gating of HERG.     

NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity.

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.     

Effect of nitration on the physicochemical and kinetic features of wild-type and monotyrosine mutants of human respiratory cytochrome c

The effect of tyrosine nitration on the physicochemical properties and reactivity of human respiratory cytochrome c has been extensively analyzed. A set of mutants, each bearing only one tyrosine out of the five present in the wild-type molecule, has been constructed in order to study the effect of each tyrosine nitration on the properties of the whole protein. Replacement of tyrosines by phenylalanines does not promote significant changes in the properties of the cytochrome. Nitration of wild-type cytochrome c promotes a drastic decrease (ca. 350 mV) in the midpoint redox potential, probably induced by nitration of both tyrosines 48 and 67. Nitration also promotes a significant decrease in the intrinsic reactivity of all the wild-type and mutant proteins. Nitration of mutant cytochromes and, in particular, of the wild-type protein significantly decreases their reactivity with cytochrome c oxidase, thereby suggesting that this alteration is due to an accumulative effect of different nitrations. The reactivity of mutants bearing tyrosine 67 and, to a lesser extent, tyrosine 74 is more affected by nitration, indicating that the change in reactivity of nitrated wild-type cytochrome c is mainly due to nitration of these tyrosine residues. Moreover, nitration of wild-type cytochrome c induces a significant loss in its ability to activate caspases because of the additive effect of nitration of several tyrosine groups, as inferred from the behavior of monotyrosine mutants.     

Hydrodynamic properties of rigid macromolecules composed of ellipsoidal and cylindrical subunits.

A procedure is devised for the calculation of hydrodynamic properties of rigid macromolecules composed subunits that are modeled as ellipsoids of revolution and cylinders. Owing to the axial symmetry of these shapes, smooth shell models can be constructured for the subunit structure. The bead shell model so constructed is employed for the calculation of the properties. A computer program, HYDROSUB, has been written implementing both the model building and the hydrodynamic calculation. A detailed example of the use of this methodology is presented for the case of the solution properties of the human antibody molecule immunoglobulin G3 (IgG3). Finally, hints are given on other uses and applications of the procedure.     

Two new Marina (Leguminosae) from the southern Baja California peninsula, Mexico

Marina victoriae andM. brevis are newly described species from the southern Baja California peninsula, Mexico. The former is a perennial herb locally abundant in the eroded hillsides at the foothills of Sierra de la Victoria, where an oak woodland and a dry tropical forest meet. The latter is an elusive ephemeral inhabiting a narrow strip of land between the mangroves and the desert scrub on an island in the Gulf of California. These two new species belong to the seriesChrysorrhizae, a group native to the Gulf of California basin.     

Chlorophyll-a determination with ethanol – a critical test

Chlorophyll-a content is widely used as an indicator of the quality of freshwater bodies. Quantification of chlorophyll-a is a routine procedure in the test laboratories of water works, and in research laboratories. Although attempts have been made to standardise the measurement procedure, there are nonetheless many procedures currently in use. This work is focused on a careful re-examination of the ISO: 10260, 1992 standard, which prescribes 90% (v/v) ethanol for chlorophyll extraction and measurement. Chlorophyll contents of cultures of the cyanobacterium Synechococcus elongatus Nägeli and the chlorophyte Scenedesmus acutus Meyen were determined by means of a series of concentrations of ethanol/water mixtures which were employed as extracting agents – the water content was gradually decreased from 20 to 0%. The extraction procedure was verified by measuring the amount of retained water after using both water and oil pumps for filtering the samples. The spectroscopic effects of the presence of water were studied and the molecular background of these spectral phenomena is discussed. The extraction yields obtained with 90% ethanol were compared to those obtained with methanol and acetone. On the basis of the calculated error level, improvements to the ISO: 10260, 1992 standard method have been suggested.     

Population growth kinetics of the nematode, Steinernema feltiae, in submerged monoxenic culture

Monoxenic cultures of the nematode, Steinernema feltiae, were carried out on two complex liquid media: P1, mainly soybean flour/egg yolk/yeast extract, and P2, mainly egg yolk/yeast extract. Up to 140 000–200 000 nematodes ml^–1 were produced within 7 days, and more than 95% of the final population was in the infective juvenile stage. The total nematode concentration growth curve had a sigmoidal shape. Nematode population growth kinetics were modelled using a re-parameterised Gompertz model. Yeast extract concentration appeared to be a key factor for obtaining high nematode concentrations.