Development of expressed sequence tag-simple sequence repeat markers for Chrysanthemum morifolium and closely related species.

With the development of chrysanthemum breeding in recent years, an increasing number of wild species in genera related to Chrysanthemum were introduced to extend the genetic resources and facilitate the genetic improvement of chrysanthemums via hybridization. However, few simple sequence repeat (SSR) markers are available for marker-assisted breeding and population genetic studies of chrysanthemum and closely related species. Expressed sequence tags (ESTs) in public databases and cross-species transferable markers are considered to be a cost-effective means for developing sequence-based markers. In this study, 25 EST-SSRs were successfully developed from Chrysanthemum EST sequences for Chrysanthemum morifolium and closely related species. In total, 4164 unigene sequences were assembled from 7180 ESTs of chrysanthemum in GenBank, which were subsequently used to screen for the presence of microsatellites with the SSRIT software. The screening criteria were 8, 5, 4, and 3 repeating units for di-, tri-, tetra-, and penta- and higher-order nucleotides, respectively. Moreover, 310 SSR loci from 296 sequences were identified, and 198 primer pairs for SSR amplification were designed with the Primer Premier 5.0 software, of which 25 SSR loci showed polymorphic amplification in 52 species and varieties belonging to Chrysanthemum, Ajania, and Opisthopappus. The application of EST-SSR markers to the identification of intergeneric hybrids between Chrysanthemum and Ajania was demonstrated. Therefore, EST-SSRs can be developed for species that lack gene sequences or ESTs by utilizing ESTs of closely related species.

Screening of molecular markers linked to dwarf trait in crape myrtle by bulked segregant analysis.

Plant height is one of the most important traits of plant architecture as it modulates both economic and ornamental values. Crape myrtle (Lagerstroemia indica L.) is a popular ornamental woody plant because of its long-lasting mid-summer bloom, rich colors, and diversified plant architecture. These traits also make it an ideal model of woody species for genetic analysis of many ornamental traits. To understand the inheritance of plant height and screen for genes modulating plant height in Lagerstroemia, segregation of the plant height trait was analyzed using the F1 population of L. fauriei (standard) x L. indica 'Pocomoke' (dwarf) with 96 seedlings, while dwarf genes were screened using the bulked segregant analysis method, combined with 28 amplified fragment length polymorphism primers and 41 simple sequence repeat primers. The results showed that the dwarf trait of crape myrtle was controlled by a major gene and modified by minor genes. An amplified fragment length polymorphism marker, M53E39-92, which was 23.33 cM from the loci controlling the dwarf trait, was screened. These results provide basic information for marker-assisted selection in Lagerstromia and cloning of dwarf genes in future studies.

Genetic diversity of Lagerstroemia (Lythraceae) species assessed by simple sequence repeat markers.

Lagerstroemia (crape myrtle) are famous ornamental plants with large pyramidal racemes, long flower duration, and diverse colors. However, little is known about the genetic structure and diversity of germplasm in Lagerstroemia. We genotyped 81 L. indica cultivars, five other species of Lagerstroemia, and 10 interspecific hybrids using 30 simple sequence repeat markers; 275 alleles were generated with a mean of nine alleles per locus. The mean polymorphism information content value, a measure of gene diversity, was 0.63, with a range from 0.25 to 0.86. The mean observed heterozygosity (0.51) tended to be lower than the mean expected heterozygosity (0.67). The mean F-statistics (F(ST), F(IS), and F(IT)) were 0.05, 0.20, and 0.24, respectively, indicating a high level of genetic variation among cultivars. Clustering analysis based on genetic distance divided the 96 genotypes into three distinct groups, which corresponded with their genetic backgrounds and geographic regions. L. indica cultivars and the other five L. species were grouped into different sub-clusters. Chinese and North American cultivars were divided into different clusters. These data about the genetic relationship among cultivars demonstrated the potential value of L. indica cultivars and other Lagerstroemia species for widening the genetic basis of breeding programs for this ornamental flower.

Determination of ceftibuten in sputum by column-switching high-performance liquid chromatography on-line with thermospray mass spectrometry.

A column-switching high-performance liquid chromatographic assay on-line with thermospray mass spectrometric detection is described for the determination of ceftibuten, an oral cephalosporin antibiotic, in human sputum. The method does not require sample pretreatment and provides increased selectivity not available from the previously reported method with UV detection. The thermospray mass spectrometric detection conditions were optimized for ceftibuten. The technique has a detection limit of 0.50 micrograms/mL and allows precise, simple, and accurate determination of ceftibuten in sputum over the range 0.50-10.00 micrograms/mL.

Determination of a cephalosporin antibiotic, ceftibuten, in human plasma with column-switching high-performance liquid chromatography with ultraviolet detection.

A column-switching high-performance liquid chromatographic assay combined with a heart-cutting technique and UV detection (LC/LC/UV) was developed to determine ceftibuten, a new oral cephalosporin, in human plasma. Plasma samples were directly injected into the first chromatographic column for sample cleanup and extraction. Thereafter, a switching valve, located at the junction of the first cleanup column and the second analytical column, opened during a 4-6-min interval after injection to transfer the segment containing ceftibuten into the analytical column for quantitation. The method was used routinely in pharmacokinetic studies and has the advantages of simplicity, improved sensitivity, and selectivity. Concentrations of ceftibuten in plasma over a range of 0.1-20 micrograms/mL can be determined with high precision and reproducibility. A single, oral, 200-mg dose of ceftibuten in humans resulted in a maximum concentration of 9.79 micrograms/mL, an area under the plot of concentration of drug in plasma versus time (0-12 h) of 47.77 micrograms.h/mL, and a half-life of 3.2 h. These results document the utility of the LC/LC/UV method in clinical pharmacokinetic studies.

Differences in the pharmacokinetics of cocaine in naive and cocaine-experienced rats.

Enhanced cocaine concentrations in brain and blood observed after an intraperitoneal challenge dose in rats exposed to cocaine for 10 days by subcutaneous administration are traced to a change in the absorption process from the site of an intraperitoneal injection to general circulation. This conclusion is reached by three sets of corroborating results: (a) Adipose tissue of rats treated for 10 days with repeat subcutaneous injections of cocaine did not reveal a buildup of cocaine in sufficient concentrations to account for the twofold increase in brain and blood concentrations seen during intraperitoneal administration; (b) administration of the drug by an intravenous route after 10-day cocaine treatment did not show a significant difference between treatment and control groups; (c) nonlinear regression on the intravenous and intraperitoneal data sets using a two-compartment open model indicated a difference in the absorption process but not in the metabolic and blood-brain transfer processes.

Extracellular concentrations of cocaine and dopamine are enhanced during chronic cocaine administration.

Chronic cocaine administration produces significant increases in cocaine-induced locomotor activity and stereotypy. In vivo microdialysis procedures were used to monitor extracellular dopamine (DA) and cocaine concentrations in the nucleus accumbens (N ACC) and cocaine concentrations in plasma of animals that received chronic or acute cocaine treatments. Following a cocaine challenge injection, concentrations of both cocaine and DA increased to significantly higher levels over time in animals that had received daily cocaine injections for 10 or 30 days than in control animals that received daily injections of saline. Concentrations of cocaine and DA in the N ACC reached maximum levels in the first 30 min following a challenge injection of cocaine. The maximum cocaine concentrations of 10- and 30-day chronic animals were, respectively, 186% and 156%, whereas the maximum DA concentrations were 264% and 216% above the maximum values observed in acute control animals. The results indicate that reverse tolerance effects observed following chronic cocaine administration may in part be accounted for by increased cocaine concentrations. Furthermore, chronic cocaine administration (over a 10- or 30-day period) increased the concentration of cocaine detected in plasma above control levels following a challenge injection. The increase in brain concentrations of cocaine in chronic animals is apparently due to increased concentrations of cocaine in plasma. A physiological change occurs in the periphery as a result of chronic cocaine administration that increases cocaine concentrations in plasma, increases extracellular cocaine levels in the brain, and increases the extracellular concentration of DA in the N ACC.

Extracellular cocaine and dopamine concentrations are linearly related in rat striatum.

Microdialysis and gas chromatography/mass spectrometry were used to determine temporal cocaine concentration profiles in the rat striatum following intraperitoneal (i.p.) cocaine injection. For a 30 mg/kg i.p. dose, cocaine reached a maximum in vivo concentration of 10.1 microM within 30 min, and then rapidly declined. A non-linear fit of a kinetic model to the experimental cocaine data gave a first-order rate constant for the appearance of cocaine in the extracellular fluid of the striatum after a 30 mg/kg dose of cocaine of 0.0304/min and a first-order rate constant of 0.0386/min for the disappearance of cocaine from the extracellular fluid. When combined with previous dopamine results for a 30 mg/kg i.p. cocaine dose, cocaine concentrations were found to be highly correlated (r = 0.963) with dopamine concentrations for the same point in time. The slope was 36.8 nM dopamine/microM cocaine and the y-intercept was 29.9 nM dopamine. Maximum dopamine and maximum cocaine concentrations were also found to be linearly related to i.p. dose of cocaine for doses of 3, 10, and 30 mg/kg.