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.      

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Characterization of disulfide bond position in proteins and sequence analysis of cystine-bridged peptides by tandem mass spectrometry.

Tandem mass spectrometry employing high-energy, collisionally activated dissociation (CAD) is shown to be a useful method for sequencing through the cystine bridge of intermolecularly disulfide-bonded peptides. A characteristic triplet of intense fragment ions is observed corresponding to cleavage through and to either side of the disulfide bridge. These fragments define the masses of the linked peptides. Fragments due to peptide chain cleavage are also observed at lower abundance in the product-ion spectra and can be sufficient to sequence both of the disulfide-linked peptides without any prior knowledge of the peptide or protein sequence. Even in cases where the peptide sequence-related product-ion yields are poor, the intensities of the disulfide cleavage ions are usually sufficient to determine the molecular weights of the component cystine-bridged peptides. In this paper we demonstrate that the high-energy CAD tandem MS approach may be used to characterize disulfide-bonded peptides directly in complex enzymatic or chemical digests of native proteins. This obviates the need for individual purification of intermolecularly disulfide-linked peptides prior to analysis. The techniques are illustrated here for synthetic, inter- and intramolecularly disulfide-linked peptides and for human transforming growth factor-alpha (des-Val-Val-TGF-alpha), a compact protein containing 48 residues and three disulfides.     

Argininosuccinic aciduria: prenatal studies in a family at risk.

We have monitored two successive pregnancies in a family which we found to be at risk for argininosuccinic aciduria. We measured argininosuccinic acid (ASA) concentrations in amniotic fluid and utilized an indirect assay of ASA lyase activity in cultured amniotic fluid cells. The assay procedure is based on the uptake of 14C from [14C]citrulline and of [3H]leucine into protein. ASA was easily measured in amniotic fluid from the first fetus at risk, whereas none was detectable in control fluids. Amniotic fluid cells cultured from this fetus had only 5.5% of control ASA lyase activity. The pregnancy was terminated, and hepatic ASA lyase activity in the fetus was shown to be about 1.3% of control values. In addition, eight fetal tissues were analyzed for ASA, and all had significant accumulation. ASA was not detected in amniotic fluid from the second fetus at risk, and ASA lyase activity in cultured cells was 80% of control activity. Enzymatic analysis of erythrocyte lysate confirmed the diagnosis of an unaffected child (ASA lyase = 46% of control) and indicated heterozygosity. Thus, we provide further evidence that argininosuccinic aciduria can be diagnosed successfully in utero by indirect assay of ASA lyase activity in cultured amniotic fluid cells. In addition, high amniotic fluid ASA concentrations provide strong adjunctive evidence for such a prenatal determination, and may prove to be sufficient for diagnosis.     

Effect of Steric and Nuclear Changes in Steroids and Triterpenoids on Sexual Reproduction in Phytophthora cactorum

The comparative biological activity of 21 naturally occurring or synthetically derived steroids, 7 tetracyclic and pentacylic triterpenoids, and antheridiol incubated with cultures of Phytophthora cactorum has been examined. There was greater dependence on precise steric features of the sterol side chain than on the extent of nuclear unsaturation in inducing oospore formation. There was no significant effect on oospore formation by changing nuclear unsaturation in ring B from Δ⁵ to Δ⁷ or to Δ^5,7. Converting the unsaturated sterol to its corresponding stanol resulted in a significant reduction in the number of oospores produced. The effectiveness of sterols bearing different side chains in inducing oospores was found to be in the following relative order: 24α-ethyl = trans-Δ²²-24α-ethyl > trans-Δ²²-24β-ethyl = 24α-E-ethylidene = 24α-methyl > 24β-methyl = trans-Δ²²-24β-methyl = 26-methyl = saturated C₇ side chain and C-20 R (17-αH, 20-αH, right-handed conformer) = cis-Δ²²-C₇ side chain and C-20 R > saturated C₇ side chain and C-20 S (17-αH, 20-βH, right-handed conformer) > no sterol = 29-hydroxyporiferasterol = 20α-hydroxycholesterol = 24ξ-hydroxy-24-vinylcholesterol. Of the sterols examined the most significant stereochemical criterion for the induction of oospore formation was absence of bulk on the front face of C-20. This follows from the observation that 20-isocholesterol and 20α-hydroxycholesterol, in which a methyl and hydroxy group, respectively, project to the front in the right handed conformation, were inactive in stimulating production of oospores. None of the triterpenoids studied induced oospore formation to any significant degree. Oospore formation was not induced by antheridiol nor 29-hydroxyporiferasterol in combination or added separately to growing cultures of P. cactorum in the concentration range 0.01 - 10.0 milligrams per liter.     

Influenza viruses: transmission between species

The only direct evidence for transmission of influenza viruses between species comes from studies on swine influenza viruses. Antigenically and genetically identical Hsw1N1 influenza viruses were isolated from pigs and man on the same farm in Wisconsin, U.S.A. The isolation of H3N2 influenza viruses from a wide range of lower animals and birds suggests that influenza viruses of man can spread to the lower orders. Under some conditions the H3N2 viruses can persist for a number of years in some species. The isolation, from aquatic birds, of a large number of influenza A viruses that possess surface proteins antigenically similar to the viruses isolated from man, pigs and horses provides indirect evidence for inter-species transmission. There is now a considerable body of evidence which suggests that influenza viruses of lower animals and birds may play a role in the origin of some of the pandemic strains of influenza A viruses. There is no direct evidence that the influenza viruses in aquatic birds are transmitted to man, but they may serve as a genetic pool from which some genes may be introduced into humans by recombination. Preliminary evidence suggests that the molecular basis of host range and virulence may be related to the RNA segments coding for one of the polymerase proteins (P3) and for the nucleoprotein (NP).     

Sodium channel inactivation in the crayfish giant axon. Must channels open before inactivating?

Experiments on sodium channel inactivation kinetics were performed on voltage-clamped crayfish giant axons. The primary goal was to investigate whether channels must open before inactivating. Voltage-clamp artifacts were minimized by the use of low-sodium solutions and full series resistance compensation, and the spatial uniformity of the currents was checked with a closely spaced pair of electrodes used to measure local current densities. For membrane potentials between -40 and +40 mV, sodium currents decay to zero with a single exponential time-course. The time constant for decay is a steep function of membrane potential. The time-course of inactivation measured with the double-pulse method is very similar to the decay of current at the same potential. Steady-state inactivation curves measured with different test pulses are identical. The time-course of double pulse inactivation shows a lag that roughly correlates with the opening of sodium channels, but detailed comparisons with the time course of the prepulse current suggest that it is not strictly necessary for channels to open before inactivating. Measurements of the potential dependence of the integral of sodium conductance area also inconsistent with the simplest cases of models in which channels must open before inactivating.