Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

Eradication of Norway Rats for Recovery of Seabird Habitat on Langara Island, British Columbia

Introduced Rattus norvegicus (Norway rats) caused the decline of Synthliboramphus antiquus (ancient murrelets) and other seabirds breeding on Langara Island (approximately 3,100 ha), British Columbia. Using funds from the litigation settlement following the Nestucca oil spill, Environment Canada eradicated Norway rats using a technique developed in New Zealand which involved dispensing wax baits containing the anticoagulant brodifacoum at 50 ppm from fixed bait stations. Bait stations were placed every 75 to 100 m on a grid over the entire island (1 station/ha). Rats removed bait for 26 days, after which crews placed baits in protective plastic bags in each bait station. Stations loaded with baits were left on the island and rechecked four times over 2 years, after which bait stations and remaining bait were removed. The eradication succeeded. No signs of rats have been detected on Langara Island and its associated islands since January 1996. No rats were trapped during 1,700 trap‐nights following the poison campaign. Incisor marks of rats were not found on apples or oil‐dipped chew‐sticks. Corvus corax (common ravens) likely suffered greater than 50% mortality from the eradication after apparently gaining access to the poison directly from bait stations and from scavenging rat carcasses. A monitoring and response system is being developed in conjunction with current users of the islands. The success on Langara Island demonstrates how the technique proven on small New Zealand islands of less than 300 ha can be effectively extrapolated to much larger islands.     

Microbial transformation of heterocyclic molecules in deep subsurface sediments

Recently attempts have been made to establish the presence and to determine the metabolic versatility of microorganisms in the terrestrial deep subsurface at the Savannah River Plant, Aiken, SC, USA. Sediment samples obtained at 20 different depths of up to 526 m were examined to determine carbon mineralization under aerobic, sulfate-reducing, and methanogenic conditions. The evolution of¹⁴CO₂ from radiolabelled glucose was observed under aerobic conditions in all sediments, whereas pyridine was transformed in 50% of the 20 sediments and indole was metabolized in 85% of the sediments. Glucose mineralization in certain sediments was comparable to that in the surface environment. Sulfate was reduced in only five sediments, and two were carbon limited. Methane production was detected in ten sediments amended with formate only after long-term incubations. The transformation of indole and pyridine was only rarely observed under sulfate-reducing conditions and was never detected in methanogenic incubations. This study provides information concerning the metabolic capability of both aerobic and anaerobic microorganisms in the deep subsurface and may prove useful in determining the feasibility of microbial decontamination of such environments.     

Defects in fruiting body development caused by Tn5 lac insertions in Myxococcus xanthus.

Mutations caused by insertions of Tn5 lac that block development are rare. At least six of the eight mutations examined appeared to be regulatory. Three of these were found to disrupt social motility, suggesting a particular importance for this function. One other occurred in a known cell-cell interaction gene, bsgA, and the remaining two were located in genes operative early in the developmental program.     

Direct localization of monoclonal antibody-binding sites on Acanthamoeba myosin-II and inhibition of filament formation by antibodies that bind to specific sites on the myosin-II tail

Electron microscopy of myosin-II molecules and filaments reacted with monoclonal antibodies demonstrates directly where the antibodies bind and shows that certain antibodies can inhibit the polymerization of myosin-II into filaments. The binding sites of seven of 23 different monoclonal antibodies were localized by platinum shadowing of myosin monomer-antibody complexes. The antibodies bind to a variety of sites on the myosin-II molecule, including the heads, the proximal end of the tail near the junction of the heads and tail, and the tip of the tail. The binding sites of eight of the 23 antibodies were also localized on myosin filaments by negative staining. Antibodies that bind to either the myosin heads or to the proximal end of the tail decorate the ends of the bipolar filaments. Some of the antibodies that bind to the tip of the myosin-II tail decorate the bare zone of the myosin-II thin filament with 14-nm periodicity. By combining the data from these electron microscope studies and the peptide mapping and competitive binding studies we have established the binding sites of 16 of 23 monoclonal antibodies. Two of the 23 antibodies block the formation of myosin-II filaments and given sufficient time, disassemble preformed myosin-II filaments. Both antibodies bind near one another at the tip of the myosin-II tail and are those that decorate the bare zone of preformed bipolar filaments with 14-nm periodicity. None of the other antibodies affect myosin filament formation, including one that binds to another site near the tip of the myosin-II tail. This demonstrates that antibodies can inhibit polymerization of myosin-II, but only when they bind to key sites on the tail of the molecule.     

The fine structure of the lateral eyes ofNeocarus texanus Chamberlin and Mulaik, 1942 (Opilioacarida,Acari, Arachnida,Chelicerata)

Summary Neocarus texanus, a primitive mite, bears two pairs of eyes, which are principally similar in ultrastructure. Each eye is covered externally by a cuticular cornea. It is underlain by flat sheath cells which send extensive processes into the retina. The retina is composed of distal and proximal cells. The 20 distal cells of the anterior eye are inversely orientated and form 10 disc-like rhabdoms. They represent typical retinula cells. Each rhabdom encloses the dendritic process of a neuron, the perikaryon of which is located outside the retina (proximal cells). The significance of this cell is not known. The retina is underlain by a crystalline tapetum. In the posterior eye 14 retinula cells form 7 rhabdoms in an arrangement similar to the anterior eye. The eyes of one side of the body are located within a capsule of pigment cells. Together the axons of the distal and proximal cells form the two optic nerves, one on each side of the body. The optic nerves leave the eyes anteriorly and terminate in two optic neuropils located in the brain.From structural evidence it is concluded, that the resolution of the eyes must be rather low.The peculiar proximal cells have not been observed previously in Acari. They probably resemble at best the eccentric cells and arhabdomeric cells of xiphosurans, scorpions, whip-scorpions and opilionids. Also, inverse retinae and tapeta of the present type have not been found in Acari until now, but are present in other Arachnida. Thus the eyes ofNeocarus texanus evidently represent a unique type within the Acari.     

Acquisition and assimilation of gaseous ammonia as revealed by intracellular pH changes in leaves of higher plants

Atmospheric ammonia (NH₃) from various anthropogenic sources has become a serious problem for natural vegetation. Ammonia not only causes changes in plant nitrogen metabolism, but also affects the acid-base balance of plants. Using the pH-sensitive fluorescent dyes pyranine and esculin, cytosolic and vacuolar pH changes were measured in leaves of C₃ and C₄ plants exposed for brief periods to concentrations of NH₃ in air ranging from 1.33 to 8.29 mol NH₃ · mol^-1 gas (0.94–5.86 mg · m^-3). After a lag phase, uptake of NH₃ from air at a rate of 200 nmol NH₃ · m ^{- 2} leaf area · s^{- 1} into leaves of Zea mays L. increased pyranine fluorescence indicating cytosolic alkalinisation. The increase was much larger in the dark than in the light. In illuminated leaves of the C₃ plant Pelargonium zonale L. and the C₄ plants Z. mays and Amaranthus caudatus L., NH₃-dependent cytosolic alkalinisation was particularly pronounced when CO₂ was supplied at very low levels (16 or 20 mol CO₂ · mol^{- 1} gas, containing 210 mmol O₂ · mol^{- 1} gas). An increase in esculin fluorescence, which was smaller than that of pyranine, was indicative of trapping of some of the NH₃ in the vacuoles of leaves of Spinacia oleracea L. and Z. mays. Photosynthesis and transpiration remained unchanged during exposure of illuminated leaves to NH₃, yielding an influx of 200 nmol NH₃ · m^-2 leaf area · s^-1 for up to 30 min, the longest exposure time used. Both CO₂ and O₂ influenced the extent of cytosolic alkalinisation. At 500 mol CO₂ · mol^-1 gas the cytosolic alkalinisation was suppressed more than at 16 or 20 mol CO₂ · mol^-1 gas. The suppressing effect of CO₂ on the NH₃induced alkalinisation was larger in illuminated leaves of the C₄ plants Z. mays and A. caudatus than in leaves of the C₃ plant P. zonale. A reduction of the O₂ concentration from 210 to 10 mmol O₂ · mol ^-1 gas, which inhibits photorespiration, increased the NH₃induced cytosolic alkalinisation in C₃ plants. Suppression by CO₂ or O₂ of the alkaline pH shift caused by the dissolution and protonation of NH₃ in queous leaf compartments, and possibly by the production of organic compounds synthesised from atmospheric NH₃, indicates that NH₃ which enters leaves is rapidly assimilated if photosynthesis or photorespiration provide nitrogen acceptor molecules.This work was supported by the Biotechnology and Biological Sciences Research Council and the Deutsche Forschungsgemein-schaft within the framework of the research of Sonderforschun-gsbreich 251 of the University of Würzburg. We are grateful to Dr. B. Wollenweber (The Royal Veterinary and Agricultural University, Denmark) for discussions.     

A kinetic study of interactions of (Rp)- and (Sp)-adenosine cyclic 3',5'-phosphorothioates with type II bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase

The stereoselectivity of the adenosine cyclic 3',5'-phosphate (cAMP) binding sites on the regulatory subunit of the type II bovine cardiac muscle cAMP-dependent protein kinase was investigated by examining the interactions of (Rp)- and (Sp)-adenosine cyclic 3',5'-phosphorothioates (cAMPS) with these sites. While activation of the holoenzyme and binding to the regulatory subunit of the type II kinase were observed for both of these diastereomers, there were significant differences between the interactions of the cAMPS isomers with the enzyme. In particular, the Sp isomer is more potent than the Rp species not only in the activation of reconstituted, as well as directly isolated, holoenzyme but also in the inhibition of [3H]cAMP binding to the regulatory subunit. A marked preference for the binding of the Sp isomer to site 2 in the regulatory subunit exists. Hydrogen bonding of a functional group on the regulatory subunit with preferential orientation toward the exocyclic oxygen rather than the sulfur of the thiophosphoryl residue may be involved in the observed selectivity of cAMPS binding and activation. In addition to our findings on the stereoselectivity of the binding of cAMPS to cAMP-dependent protein kinase, we have established a method for the reconstitution of holoenzyme from the purified subunits without subjecting the regulatory protein to denaturing conditions.     

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment

DNA flow histogram analysis, using 33342 Hoechst as a stain, has been used to detect the effect of the potentially bifunctional alkylating agent, mitomycin C (MMC) on dermal fibroblasts from patients with Fanconi's anemia (FA), a hereditary human disease characterized by pancytopenia, hypersensitivity to DNA-crosslinking agents, congenital abnormalities and a predisposition for neoplasia. At 24 or 48 hr after a 2-hr exposure to 0.05 or 0.10 micrograms/ml MMC, (3)HdT incorporation was reduced to a greater extent in FA cells than in normal cells. Cells sorted from the last half of S phase showed a slightly greater inhibition of (3)HdT incorporation than did those sorted from the first half of S. Fanconi's anemia cells exhibited a marked accumulation in the G(2) + M peak of flow histograms following exposure to MMC. Twenty-four hr after treatment with .0.5 micrograms/ml MMC, the G(2) + M fraction of FA cells (eight lines) increased to more than 0.5 from a control value of approximately 0.02. Both normals (six lines) and heterozygotes (eight lines) showed, on the average, much less of a G(2) + M increment than did FA cells, even after exposure to 0.1 micrograms/ml MMC. Examination of cells sorted from the G(2) + M peak revealed that MMC-treated FA cells were blocked prior to mitosis. To determine whether the response of FA cells was specific for bifunctional alkylating agent, cells were also treated with ethylmethanesulfonate, a monofunctional agent. Twenty-four hours after exposure to 0.25 or 0.5 mg/ml ethylmethanesulfonate, FA and normal cells showed similar, small increases in the G(2) + M peak. The results suggest the utility of flow cytometry in the diagnostic evaluation of fibroblasts from patients suspected of having Fanconi's anemia.