[Show abstract][Hide abstract] ABSTRACT: Phylogeographic inference can determine the timing of population divergence, historical demographic processes, patterns of migration, and when extended to multiple species, the history of communities. Single-locus analyses can mislead interpretations of the evolutionary history of taxa and comparative analyses. It is therefore important to revisit previous single-locus phylogeographic studies, particularly those that have been used to propose general patterns for regional biotas and the processes responsible for generating inferred patterns. Here, we employ a multilocus statistical approach to re-examine the phylogeography of Lampropeltis zonata. Using nonparametic and Bayesian species delimitation, we determined that there are two well-supported species within L. zonata. Ecological niche modelling supports the delimitation of these taxa, suggesting that the two species inhabit distinct climatic environments. Gene flow between the two taxa is low and appears to occur unidirectionally. Further, our data suggest that gene flow was mediated by females, a rare pattern in snakes. In contrast to previous analyses, we determined that the divergence between the two lineages occurred in the late Pliocene (c. 2.07 Ma). Spatially and temporally, the divergence of these lineages is associated with the inundation of central California by the Monterey Bay. The effective population sizes of the two species appear to have been unaffected by Pleistocene glaciation. Our increased sampling of loci for L. zonata, combined with previously published multilocus analyses of other sympatric species, suggests that previous conclusions reached by comparative phylogeographic studies conducted within the California Floristic Province should be reassessed.
[Show abstract][Hide abstract] ABSTRACT: The phylogeography of the California mountain kingsnake, Lampropeltis zonata, was studied using mitochondrial DNA sequences from specimens belonging to the seven recognized subspecies and collected throughout the range of the species. Maximum parsimony and maximum likelihood methods identified a basal split within L. zonata that corresponds to southern and northern segments of its distribution. The southern clade is composed of populations from southern California (USA) and northern Baja California, Mexico. The northern clade is divided into two subclades, a 'coastal' subclade, consisting of populations from the central coast of California and the southern Sierra Nevada Mountains of eastern California, and a 'northeastern' subclade, mainly comprised of populations north of the San Francisco Bay and from the majority of the Sierra Nevada. We suggest that past inland seaways in southwestern California and the embayment of central California constituted barriers to gene flow that resulted in the two deepest divergences within L. zonata. Throughout its evolutionary history, the northern clade apparently has undergone instances of range contraction, isolation, differentiation, and then expansion and secondary contact. Examination of colour pattern variation in 321 living and preserved specimens indicated that the two main colour pattern characters used to define the subspecies of L. zonata are so variable that they cannot be reliably used to differentiate taxonomic units within this complex, which calls into question the recognition of seven geographical races of this snake.
[Show abstract][Hide abstract] ABSTRACT: Liver mitochondrial low-Km aldehyde dehydrogenase (ALDH2, EC 18.104.22.168), the isoform responsible for the conversion of acetaldehyde to acetate, is inhibited by the sulfoxide bioactivation products of Et2NC(O)SMe (from the alcohol aversion drug disulfiram), Pr2NC(O)SEt (the herbicide S-ethyl N,N-dipropylthiocarbamate), and BuNHC(O)SMe (from the fungicide benomyl). This study tested the hypothesis that bioactivated BuNHC(O)SMe, the most potent of these thiocarbamates, is a selective carbamoylating agent for ALDH2 of mouse liver in vivo and in vitro. [14C]BuNHC(O)SMe administered i.p. to mice labeled one principal mitochondrial protein, which cochromatographed with ALDH activity by in-gel assay after isoelectric focusing. The labeled protein was isolated by isoelectric focusing (pI 6.1) and SDS-PAGE (54 kDa) and identified as ALDH2 by sequencing of peptides from a tryptic digest. In vivo at 1.5 mg/kg, enzyme inhibition was 80%, and ALDH2 was the only mitochondrial protein labeled extensively, illustrating the outstanding potency and specificity. ALDH2 also was labeled upon incubation of mouse liver mitochondria with [14C]BuNH-C(O)SMe in the presence of microsomes (P450) and NADPH. In contrast, under similar conditions, [14C]Pr2NC(O)SEt sulfoxide labeled primarily two other proteins at approximately 58 and approximately 61 kDa, establishing a very different selectivity for the two sulfoxides. These findings are of interest relative to selective inhibitors and carbamoylating agents for ALDH2 and to alcohol aversion upon exposure to herbicides and fungicides.
[Show abstract][Hide abstract] ABSTRACT: Benomyl (a non-thio fungicide) inhibits hepatic mitochondrial low-Km aldehyde dehydrogenase (mALDH or ALDH2) in ip-treated mice by 50% (IC50) at 7.0 mg/kg, which is surprisingly the same potency range as that for several dithiocarbamate fungicides (and the related alcohol abuse drug disulfiram) and thiocarbamate herbicides previously known for their alcohol-sensitizing action. The mechanism by which benomyl inhibits mALDH was therefore examined, first by comparing the metabolism of benomyl with the aforementioned mono- and dithiocarbamates and second by evaluating the inhibitory potency of the benomyl metabolites. Benomyl in ip-treated mice is converted, via butyl isocyanate, S-(N-butylcarbamoyl)glutathione, and S-(N-butylcarbamoyl)cysteine, to S-methyl N-butylthiocarbamate (MBT), identified as a transient metabolite in liver. MBT is >10-fold more potent than benomyl or butyl isocyanate as an in vivo mALDH inhibitor and is also more potent than the intermediary S-(N-butylcarbamoyl) conjugates. Benomyl and MBT inhibit mouse hepatic mALDH in vitro with IC50s of 0.77 and 8.7 microM, respectively. The potency of MBT is greatly enhanced by fortification of the mitochondria with NADPH alone or plus microsomes giving IC50s of 0.50 and 0.23 microM, respectively. This activation of MBT is almost completely blocked by the cytochrome P450 inhibitor N-benzylimidazole but not by several other cytochrome P450 inactivators. MBT (probably following bioactivation) inhibits mALDH in vivo with an IC50 of 0.3 mg/kg. Two candidate activation products were synthesized for potency determinations. N-Hydroxy MBT (prepared via the trimethylsilyl derivative) was not detected as an MBT metabolite; its low potency also rules against N-hydroxylation as the activation process. MBT sulfoxide, from oxidation of MBT with magnesium monoperoxyphthalate in water, is one of the most potent inhibitors known for mALDH and yeast ALDH in vitro (IC50 0.08-0.09 microM). These findings are consistent with a six-step bioactivation of benomyl, via the metabolites above and N-butylthiocarbamic acid, with MBT as the penultimate and MBT sulfoxide as the ultimate inhibitor of mALDH.
Chemical Research in Toxicology 05/1998; 11(5):535-43. · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: S-Methylation is a new bioactivation mechanism for metam and metabolites of methyl isothiocyanate and dazomet in mice. These soil fumigants are converted to S-methyl metam [MeNHC(S)SMe] which reaches peak levels in liver, kidney, brain, and blood 10-20 min after intraperitoneal (ip) treatment. The half-life of S-methyl metam administered ip is 8-12 min in each of these tissues. S-Methyl metam-oxon [MeNHC(O)SMe] is also detected as a metabolite of each of these soil fumigants on analysis by gas chromatography/mass spectrometry with chemical ionization. The conversion of methyl isothiocyanate to S-methyl metam and its oxon probably involves conjugation with glutathione, hydrolysis to S-(N-methylthiocarbamoyl)-cysteine, cleavage by cysteine conjugate beta-lyase to release metam, and finally methylation and oxidative desulfuration. Metam and dazomet are converted to S-methyl metam by mouse liver microsomes on fortification with S-adenosylmethionine. Metam, methyl isothiocyanate, dazomet, and three metabolites (metam-oxon [MeNHC(O)SH], MeNHC(S)SMe, and MeNHC-(O)SMe) administered ip to mice at 40 mg/kg inhibit low-Km liver mitochondrial aldehyde dehydrogenase and elevate ethanol-dependent blood and brain acetaldehyde levels. Several fungicides including the dialkyldithiocarbamates as the disulfide (thiram and the related alcohol-abuse drug disulfiram) and metal salts (ziram) also yield S-methyl thiocarbamate metabolites. Eight S-alkyl and S-(chloroallyl) thiocarbamate herbicides (EPTC, molinate, butylate, vernolate, pebulate, diallate, sulfallate, and triallate), but not their S-chlorobenzyl analog (thiobencarb), undergo sequential liberation of the thiocarbamic acid and then S-methylation, forming the S-methyl thiocarbamates which are new metabolites and potential aldehyde dehydrogenase inhibitors. The S-methyl mono- and dithiocarbamate metabolites of these herbicides and fungicides are easily identified by retention time on gas chromatography and by mass spectrometry giving [MH]+ plus [R1R2NCO]+ or [R1R2NCS]+, respectively, as the two major ions.
Chemical Research in Toxicology 01/1996; 8(8):1063-9. · 3.67 Impact Factor