Study of dried blood spots technique for the determination of dextromethorphan and its metabolite dextrorphan in human whole blood by LC–MS/MS

Dried blood spots (DBSs) technology was evaluated in an assay for the quantitation of dextromethorphan (DM) and its metabolite, dextrorphan (DT), in human whole blood using high performance liquid chromatography with tandem mass spectrometry method (LC–MS/MS). Both the parent drug and metabolite were spiked in the blood matrix and subsequently allowed to dry on a specimen collection card. The dried blood spots were removed using a manual punch and then extracted into methyl tert-butyl ether (MTBE). The organic supernatant was transferred and evaporated and the residue was reconstituted in 20% acetonitrile. The overall method recovery of DM and DT was 87.8% and 95.4%, respectively. The assay was linear over the concentration range of 0.2–200 ng/mL for both analytes. Several factors that potentially affect DBS assay quantitation were investigated, such as punch size, DBS sample punch-out location, and the volume of the blood sample pipetted on the specimen collection cards. The study determined that punch size does not affect assay quantitation accuracy. Indeed, a larger punch size increases the sensitivity due to the larger sampled blood spots. Sampling from different location on the specimen collection cards shows no significant variation for both drugs. The study also shows that acceptable results can be achieved with some variation of the sample volume, which allows a simple blood sampling procedure at the test sites. To achieve the similar lower limit of quantitation (LLOQ) as the plasma assay, several blood spots at the same concentration level were stacked together and extracted. Bioanalytical assays using the DBS technique are promising given the advantages of the method over the plasma assay.     

The mating system of the Sichuan snub‐nosed monkey (Rhinopithecus roxellana)

This article reports the first genetic study of the mating system of the Sichuan snub‐nosed monkey (Rhinopithecus roxellana), an endemic and endangered species in China. The investigation was carried out in a population (WRT) in the Qinling Mountains using data from both field observation and paternity analysis through microsatellite DNA profiling. During a mating season, a male on an average copulated with 5.7 females. Approximately 18% of the females were observed to copulate with more than one male over the study period. The majority of copulations (94.5%) were initiated by females. Twenty‐eight of 430 observed matings were extra‐unit copulations. Eight polymorphic microsatellite loci were used for paternity analysis. The number of alleles at each locus ranged from 3 to 7 (mean=4.3). Observed heterozygosity ranged between 0.32 and 0.79. None of the loci showed significant deviation from Hardy–Weinberg equilibrium. Results from paternity exclusion showed that 12 of 21 (57.1%) immature individuals were sired by extra‐unit males. Although the basic social unit of snub‐nosed monkeys is consistent with a polygynous mating system, both field observation and genetic data suggests that their mating system is polygamous. Infanticide and inbreeding avoidance are the most likely explanations for the promiscuity of female snub‐nosed monkeys. Am. J. Primatol. 72:25–32, 2010. © 2009 Wiley‐Liss, Inc.     

Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair

Fanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling DNA interstrand crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe the FA protein FANCI, identified as an ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and, together, as the FANCI-FANCD2 (ID) complex, localize to chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.     

Characterization of an Escherichia coli O157:H7 plasmid O157 deletion mutant and its survival and persistence in cattle

Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic DeltapO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the DeltapO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the DeltapO157 mutant. The DeltapO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the DeltapO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the DeltapO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed approximately 50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.     

Characterization of a Bifidobacterium longum BORI dipeptidase belonging to the U34 family

A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50 degrees C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a beta-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.     

Synthesis, DNA-binding and photocleavage studies of cobalt(III) mixed-polypyridyl complexes: [Co(phen)(2)(dpta)](3+) and [Co(phen)(2)(amtp)](3+)

Two novel cobalt(III) mixed-polypyridyl complexes [Co(phen)(2)(dpta)](3+) and [Co(phen)(2)(amtp)](3+) (phen=1,10-phenanthroline, dpta=dipyrido-[3,2-a;2',3'-c]- thien-[3,4-c]azine, amtp=3-amino-1,2,4-triazino[5,6-f]1,10-phenanthroline) have been synthesized and characterized. The interaction of these complexes with calf thymus DNA was investigated by spectroscopic, cyclic voltammetry, and viscosity measurements. Results suggest that the two complexes bind to DNA via an intercalative mode. Moreover, these Co(III) complexes have been found to promote the photocleavage of plasmid DNA pBR322 under irradiation at 365nm. The mechanism studies reveal that hydroxyl radical (OH()) is likely to be the reactive species responsible for the cleavage of plasmid DNA by [Co(phen)(2)(dpta)](3+) and superoxide anion radical (O(2)(-)) acts as the key role in the cleavage reaction of plasmid DNA by [Co(phen)(2)(amtp)](3+).     

Effects of mating rates on oviposition,sex ratio and longevity in a predatory mite <Emphasis Type="Italic">Neoseiulus cucumeris</Emphasis> (Acari: Phytoseiidae)

Two aspects of mating effects on the fecundity, sex ratio and longevity of Neoseiulus cucumeris (Acari: Phytoseiidae) were examined in laboratory experiments: (1) females mated by one, two or three different males (unmated and 3 days old) at 5-day intervals, and (2) females mated by males with different age/mating status (number of females mated previously by the male). Females allowed to mate with a second or third male at 5-day intervals produced 39 eggs on average, but those mated with a single male produced 28 eggs on average. Matings with additional males 5 or 10 days after the first male increased the duration of the oviposition period of these females by 5–7 days and at the same time reduced the post-oviposition period by about 10 days. Overall, females with additional matings by one or two different males at 5-day intervals survived a few days shorter than females without additional males. Mating with a different female each day, a male of N. cucumeris could mate with 5–8 females, which produced a total of 85–116 eggs: females mated with a male during days 1 and 2 in its adulthood and with a male of the last 2 days of life (days 7 and 8) produced about half as many eggs as females mated with a male during 3–6 days of its adulthood. Females mated with males that are too young or too old had a shorter oviposition period and a longer post-oviposition period and longevity than females mated with middle-aged males. In both experiments, rates of oviposition remained similar in females with high or low fecundity. This indicates that in both cases, the increased fecundity is due to the extension of the oviposition period through additional sperm supplied by the second male and or third male (in experiment 1) or more sperm by males not too young nor too old (experiment 2).