Chloroplast evidence for geographic stasis of the Australian bird-dispersed shrub Tasmannia lanceolata (Winteraceae).

Few chloroplast-based genetic studies have been undertaken for plants of mesic temperate forests in the southern hemisphere and fossil-based models have provided evidence of vegetation history only at the broadest scales in this region. This study investigates the chloroplast DNA phylogeography of Tasmannia lanceolata (Winteraceae), a fleshy-fruited, bird-dispersed shrub that is widespread in the mountains of southeastern Australia and Tasmania. Thirty haplotypes were identified after sequencing 3206 bp of chloroplast DNA in each of 244 individuals collected across the species' range. These haplotypes showed unexpectedly strong phylogeographic structuring, including a phylogeographic break within a continuous part of the species' range, with the distribution of four major clades mostly not overlapping, and geographic structuring of haplotypes within these clades. This strong geographic patterning of chloroplast DNA provided evidence for the survival of T. lanceolata in multiple putative wet forest refugia as well as evidence for additional wet forest species refugia in southeastern Australia. In western Tasmania lower haplotype diversity below the LGM tree line compared to above the LGM tree line suggests that glacial refugia at high altitudes may have been important for T. lanceolata. The level of geographic structuring in T. lanceolata is similar to gravity dispersed southern hemisphere plants such as Nothofagus and Eucalyptus. Behavioural traits of the birds transporting seed may have had a strong bearing on the limited transport of T. lanceolata seed, although factors limiting establishment, possibly including selection, may also have been important.

Recurrent nuclear DNA introgression accompanies chloroplast DNA exchange between two eucalypt species.

Numerous studies within plant genera have found geographically structured sharing of chloroplast (cp) DNA among sympatric species, consistent with introgressive hybridization. Current research is aimed at understanding the extent, direction and significance of nuclear (nr) DNA exchange that accompanies putative cpDNA exchange. Eucalyptus is a complex tree genus for which cpDNA sharing has been established between multiple species. Prior phylogeographic analysis has indicated cpDNA introgression into the widespread forest species Eucalyptus globulus from its rare congener E. cordata. In this study, we use AFLP markers to characterize corresponding nrDNA introgression, on both a broad and fine spatial scale. Using 388 samples we examine (i) the fine-scale spatial structure of cp and nrDNA introgression from E. cordata into E. globulus at a site in natural forest and (ii) broad-scale patterns of AFLP marker introgression at six additional mixed populations. We show that while E. globulus and E. cordata retain strongly differentiated nuclear gene pools overall, leakage of nrDNA occurs at mixed populations, with some AFLP markers being transferred to E. globulus recurrently at different sites. On the fine scale, different AFLP fragments show varying distances of introgression into E. globulus, while introgression of cpDNA is extensive. The frequency of E. cordata markers in E. globulus is correlated with spatial proximity to E. cordata, but departs from expectations based on AFLP marker frequency in E. cordata, indicating that selection may be governing the persistence of introgressed fragments in E. globulus.

Maternal inheritance of mitochondria in Eucalyptus globulus.

It is important to verify mitochondrial inheritance in plant species in which mitochondrial DNA (mtDNA) will be used as a source of molecular markers. We used a polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) approach to amplify mitochondrial introns from subunits 1, 4, 5, and 7 of NADH dehydrogenase (nad) and cytochrome oxidase subunit II (cox2) in Eucalyptus globulus. PCR fragments were then either sequenced or cut with restriction enzymes to reveal polymorphism. Sequencing cox2 showed that eucalypts lack the intron between exons 1 and 2. One polymorphism was found in intron 2-3 of nad7 following restriction digests with HphI. Fifty-four F1 progeny from seven families with parents distinguishable in their mitochondrial nad7 were screened to show that mitochondria were maternally inherited in E. globulus. These results constitute the first report of mitochondrial inheritance in the family Myrtaceae.

Chloroplast sharing in the Tasmanian eucalypts.

The biogeographic pattern of chloroplast DNA (cpDNA) haplotypes in Eucalyptus on the island of Tasmania is consistent with reticulate evolution, involving at least 12 Tasmanian species from the subgenus Symphyomyrtus. Intraspecific cpDNA polymorphism in 14 of 17 species is coupled with extensive sharing of identical haplotypes across populations of different species in the same geographic area. Haplotype diversity is lowest in central regions of Tasmania formerly occupied by alpine vegetation during glacial intervals and in northern regions that were periodically linked to continental Australia by land bridges. The observed distribution of several cpDNA haplotypes unique to Tasmania coincides with modeled locations of glacial refugia in coastal areas of Tasmania and shows the power of cpDNA in unraveling the complex history of past distributions of Eucalyptus. The results suggest that the model of evolution of the eucalypts should be reassessed to allow for the anastomosing effects of interspecific hybridization and introgression.

Incongruence between chloroplast and species phylogenies in Eucalyptus subgenus Monocalyptus (Myrtaceae).

Seventy-eight polymorphic cpDNA (chloroplast DNA) characters were found in 13 closely related taxa from Eucalyptus series Amygdalinae (subgenus Monocalyptus) and seven potential outgroup taxa. The strict consensus of six cladograms generated from cpDNA data confirmed monophyly of Monocalyptus. However, cpDNA phylogeny within Monocalyptus was incongruent with taxonomic classification, being more related to geography, even when accessions were from divergent series. Monocalyptus cpDNA formed two major clades. On the island of Tasmania cpDNA was restricted to a single clade, exhibited very little variation, and was phylogenetically related to cpDNA found in central and western Victoria. In contrast, cpDNA of mainland monocalypt taxa was more variable, even within the Amygdalinae. Four out of six Tasmanian Amygdalinae species were polymorphic. The difference between cpDNA of replicates was often greater than differences between species from different series. The low level of cpDNA variation and extensive morphological intergradation between the Tasmanian endemics suggest recent speciation. However, the transfer of cpDNA through hybridization between lineages is the most likely explanation for the observed sharing of cpDNA across series. This study highlights that the geographical pattern to cpDNA variation in Eucalyptus may be an important source of information on past plant distributions in Australia.

ITS sequence data resolve higher level relationships among the eucalypts.

Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA were obtained for 35 species of Eucalyptus s.s. and seven taxa representing five outgroup genera (Allosyncarpia, Angophora, Arillastrum, Corymbia, and Stockwellia). The sequences were analyzed cladistically. The data distinguished clearly between the two major subgenera of Eucalyptus s.s. (Symphyomyrtus and Monocalyptus) but indicated that subgenus Eudesmia may be paraphyletic. ITS sequence data demonstrated the potential to resolve relationships between sections within subgenus Symphyomyrtus. Within sections, however, taxa were poorly differentiated. At the generic level, Corymbia appeared to be paraphyletic due to the exclusion of Angophora. The positions of Allosyncarpia and Arillastrum relative to the ingroup remain unresolved. ITS sequence data may prove valuable for resolving other phylogenetic relationships at higher taxonomic levels within Eucalyptus.

Studies on the renal excretion of the acyl glucuronide, phenolic glucuronide and sulphate conjugates of diflunisal.

1. In five healthy male volunteers given multiple doses of diflunisal (DF), renal clearances (CLR) of the acyl glucuronide (DAG), phenolic glucuronide (DPG) and sulphate (DS) conjugates were about 42, 25 and 13 ml min-1, respectively. 2. These relatively low CLR values are probably due largely to the very high plasma protein binding of the conjugates, found in vitro to be 99.0%, 97.8% and 99.45%, respectively. 3. Thus glomerular filtration plays the minor and active tubular secretion the major role in renal excretion of the three conjugates. 4. This conclusion was supported by the effect of probenecid co-administration, which decreased CLR of DAG and DPG by about 70%. CLR for DS could not be calculated when probenecid was co-administered (because of interference by probenecid metabolites in the analysis of DS in urine). 5. Water-induced diuresis had no effect on CLR of the DF conjugates, consistent with tubular reabsorption being negligible.

Gas chromatographic assay of vigabatrin enantiomers in plasma.

A rapid and specific gas chromatographic method has been developed for the determination of plasma R(-)- and S(+)-vigabatrin concentrations. The method involves a double derivatisation step, chromatography on a megabore Chirasil-Val capillary column and thermionic specific detection. Concentrations in the range 1.0-200 micrograms/ml can be measured for R(-)-vigabatrin, and 0.5-100 micrograms/ml for S(+)-vigabatrin, using 250 microliters of plasma. The assay is suitable for pharmacokinetic studies and routine therapeutic drug monitoring in humans.

Metabolism of diazepam and related benzodiazepines by human liver microsomes.

The metabolism of diazepam has been studied in vitro using microsomal preparations from five human livers. An HPLC method was developed for the assay of diazepam, its congeners and its metabolites. Various methods for the incorporation of diazepam into the incubation medium were explored. It was shown that the use of organic solvents or small quantities of hydrochloric acid enhanced the solubility of this substrate. However all of the organic solvents tested were associated with substantial (around 50%) inhibition of metabolism of diazepam by both major pathways (N-demethylation and C3-hydroxylation). The use of hydrochloric acid gave satisfactory solubilization of diazepam, but not of pinazepam, prazepam or halazepam. Detailed metabolic studies were conducted only for diazepam, using neither hydrochloric acid nor organic solvents in the incubation medium. Formation of N-desmethyl-diazepam increased approximately linearly with diazepam concentration to 200 microM, and did not show saturation. Formation of temazepam gave a curved profile over the same range of diazepam concentrations, suggestive of a sigmoidal relationship. Michaelis-Menten parameters could not be determined for either reaction, but intrinsic clearances for N-demethylation varied over a 6-fold range. Diazepam N-demethylation was apparently promoted by the inclusion of temazepam in the incubation medium, while C3-hydroxylation of diazepam was enhanced in the presence of N-desmethyldiazepam. Mephenytoin in the incubation mixture had no effect on diazepam metabolism by either pathway. The present studies have defined some of the methodological problems inherent in in vitro metabolic studies with benzodiazepines, and have shed further light on the metabolism of diazepam in vitro by human liver.

Phenytoin metabolism during pregnancy.

The steady-state 72 h urinary excretion of various phenytoin metabolites has been measured in 10 epileptic women, whose plasma phenytoin concentrations relative to the phenytoin dose fell during pregnancy and rose again post-partum. In later pregnancy and post partum, a mean of 61.3% and 48.9%, respectively, of the total daily phenytoin dose was eliminated as 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH). Even though p-HPPH accounts for not much more than half the total daily phenytoin dose, increased excretion of this metabolite sufficed to account for the elimination of the entire increase in the dose of phenytoin required during pregnancy. There was no definite increase in the excretion of any other (minor) metabolite measured. Thus pregnancy seems not to enhance uniformly the capacity of the various metabolic pathways of phenytoin.

The influence of other anticonvulsants on the plasma concentration of E-2-en-valproate.

E-2-en-valproate is a major metabolite present in the blood of humans treated with valproate. In animals it is a potent anticonvulsant. We have measured concentrations of valproate and E-2-en-valproate in 102 plasma samples obtained from 75 adult patients (20 taking valproate only; 55 taking valproate and other anticonvulsants) under steady-state conditions. The two groups' mean ages and weights were comparable. The average valproate daily dose was lower (p < 0.002) in the monotherapy group (1152 +/- S.D. 661 mg/d) than in the polypharmacy group (1902 +/- S.D. 874 mg/d). Despite this, the mean plasma levels of valproate and E-2-en-valproate were significantly higher (p < 0.05, p < 0.0001, respectively) in the monotherapy group (60.0 +/- S.D. 22.6 micrograms/ml; 3.00 +/- S.D. 1.40 micrograms/ml, respectively) than in the polypharmacy group (49.5 +/- S.D. 24.8 micrograms/ml; 1.73 +/- S.D. 0.95 microgram/ml). While the mean plasma valproate level was 17.5% lower in the polypharmacy group, the mean plasma E-2-en-valproate level was 42% lower. The co-administration of other anticonvulsants significantly reduced the concentration of valproate and, more so, of E-2-en-valproate in plasma.

Valproate metabolism during hepatotoxicity associated with the drug.

Plasma concentrations of valproate and certain of its metabolites and their patterns of excretion in urine are described in three adults who developed hepatotoxicity during treatment of epilepsy with sodium valproate. One patient also developed a degree of reversible renal insufficiency, whilst another may have had associated infectious mononucleosis. All three cases showed evidence of impaired mitochondrial beta-oxidation of valproate. In one the impairment was at the stage catalysed by fatty acyl-CoA dehydrogenase, in another at the stage catalysed by 3-hydroxyacyl-CoA dehydrogenase and in the third at the stage catalysed by enoyl-CoA hydratase and possibly also at the next stage catalysed by 3-hydroxyacyl-CoA dehydrogenase. The impaired beta-oxidation meant that valproate metabolism was diverted into various alternative pathways. Plasma concentrations of the suspected hepatotoxic metabolite 4-en-valproate were normal for the valproate-treated population in all cases. By analogy with certain spontaneous and acquired human disorders of branched chain amino acid metabolism, it is suggested that valproate-associated hepatotoxicity may represent the consequences of a valproate overload on a limited mitochondrial beta-oxidation capacity, causing accumulation of a toxic product of endogenous branched chain amino acid metabolism.

Covalent binding of diflunisal and probenecid to plasma protein in humans: persistence of the adducts in the circulation.

Acyl glucuronide conjugates of carboxylic drugs have been shown over the past decade to be potentially reactive metabolites, undergoing hydrolysis, intramolecular rearrangement and intermolecular transacylation reactions. The present report describes the covalent binding of diflunisal (D) and probenecid (P) to plasma protein of five healthy volunteers who took a 6-day course of oral D with oral P co-administered during the last 2 days. Maximum concentrations of the D- and P-adducts were achieved within one day of cessation of dosing, and were 35 +/- SE 1 and 17 +/- SE 1 ng/mg protein respectively. The D-protein adduct was eliminated from plasma in a biphasic manner, with a terminal half-life of 10.0 +/- SE 0.9 days. In contrast, elimination of the P-protein adduct was monophasic with a half-life of 13.5 +/- SE 0.3 days. The adducts were still measurable in plasma at least one month after the parent reversibly-bound drugs were undetectable. It is not known whether such covalent binding of the drugs, presumably via their acyl glucuronides, could have any biological consequences, such as induction of hypersensitivity reactions.

Simultaneous quantitation of salivary carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbitone by high-performance liquid chromatography.

A rapid, sensitive and accurate high-performance liquid chromatographic method for the simultaneous quantitation of phenobarbitone, phenytoin, carbamazepine and carbamazepine-10,11-epoxide in saliva is described. Only small volumes of saliva (100 microliters) are required. Separation of the drugs is achieved by reversed-phase chromatography on a Nova-Pak C18 column, with a mobile phase of acetonitrile-phosphate buffer at a flow-rate of 2.0 ml/min. Detection is effected by ultra-violet absorption at 215 nm. The total run time is under 12.5 min per assay. A precipitation but no extraction step is involved, simplifying the assay method. Salivary concentrations in the range 0.25-25 micrograms/ml for carbamazepine, 0.5-20 micrograms/ml for phenytoin and phenobarbitone and 0.4-20 micrograms/ml for carbamazepine-10,11-epoxide can be measured. Recovery varies from 94 to 108%. The method has been used for routine measurements of anticonvulsants in saliva collected daily from patients with intractable epilepsy.