Measurement of epidoxorubicin and its metabolites by high-performance liquid chromatography using an advanced automated sample processor

A sensitive and rapid method for measuring epidoxorubicin and its six metabolites by high-performance liquid chromatography using an advanced automated sample processor is described. Plasma samples (1 ml) were extracted using C₂ cassettes, and reversed-phase chromatography was performed with an Apex II ODS column. The isocratic mobile phase of acetonitrile—0.019 M NaH₂PO₄ (pH 4.0) had a flow-rate of 1 ml/min and the fluorescence detector an excitation wavelength of 480 nm with an emission at 580 nm. Linear calibration curves were obtained which were reproducible both within-day and day-to-day (coefficients of variation < 10%). The extraction efficacy of epidoxorubicin was 88% and ranged from 51 to 88% for the metabolites. This method has been successfully applied to measure the plasma levels of these compounds in patients receiving epidoxorubicin over a wide dose range (12–120 mg/m²) and in patients with disturbed liver biochemistry.     

Comparison of three techniques for administering radiolabeled substrates to sediments for trophic studies: Incorporation by microbes

Three principal methods have been used to administer substrates to sediments: injection, porewater replacement, and slurry. Here we assess how each of these techniques affects incorporation of radiolabels into macromolecules of marine sedimentary microbes. Eighty-five cores of intertidal sand were collected in a randomized-block, factorial design. One set of cores received¹⁴C-bicarbonate/³H-thymidine and was incubated in the light; another set received¹⁴C-acetate/³H-thymidine and was incubated in the dark. Following a 5-hour incubation, sediments were analyzed for incorporation of radiolabel into lipid fractions (neutral, glyco-, and polar) and DNA. The three methods of isotope administration were also applied to cores subsequently analyzed for polar lipid phosphates and phospholipid fatty-acid (PLFA) profiles. In general, incorporation was greatest when injections were made, consistent with the prediction that incorporation would decrease as specific activity of the radiolabeled substrate was diminished by dilution. The ratio of¹⁴C from acetate incorporated into polar and glycolipid fractions indicated that a significant disturbance accompanied the porewater and slurry techniques. Substantial amounts of³H were recovered in the neutral-lipid fraction, indicating that thymidine was catabolized by sedimentary microbes and tritiated products were incorporated by eukaryotes. There were no significant differences in PLFA profiles or estimates of microbial biomass among methods or controls. Incorporation of³H into DNA was similar with all combinations of methods and radiocarbon substrates.¹⁴C was extensively incorporated into DNA, indicating that photoautotrophs and heterotrophs utilized radiocarbon from bicarbonate and acetate, respectively, for de novo synthesis of DNA. Injection is suggested as the method of choice, as it presents more flexibility in its application than porewater replacement and disturbs the consortia of gradients in sediments to a significantly lesser degree than porewater replacement and slurry.     

Evolution of the CD163 family and its relationship to the bovine gamma delta T cell co-receptor WC1

Background  

The scavenger receptor cysteine rich (SRCR) domain is an ancient and conserved protein domain. CD163 and WC1 molecules are classed together as group B SRCR superfamily members, along with Spα, CD5 and CD6, all of which are expressed by immune system cells. There are three known types of CD163 molecules in mammals, CD163A (M130, coded for by CD163), CD163b (M160, coded for by CD163L1) and CD163c-α (CD163L1 or SCART), while their nearest relative, WC1, is encoded by a multigene family so far identified in the artiodactyl species of cattle, sheep, and pigs.     

Characterization of functional domains of equine infectious anemia virus Rev suggests a bipartite RNA-binding domain

Equine infectious anemia virus (EIAV) Rev is an essential regulatory protein that facilitates expression of viral mRNAs encoding structural proteins and genomic RNA and regulates alternative splicing of the bicistronic tat/rev mRNA. EIAV Rev is characterized by a high rate of genetic variation in vivo, and changes in Rev genotype and phenotype have been shown to coincide with changes in clinical disease. To better understand how genetic variation alters Rev phenotype, we undertook deletion and mutational analyses to map functional domains and to identify specific motifs that are essential for EIAV Rev activity. All functional domains are contained within the second exon of EIAV Rev. The overall organization of domains within Rev exon 2 includes a nuclear export signal, a large central region required for RNA binding, a nonessential region, and a C-terminal region required for both nuclear localization and RNA binding. Subcellular localization of green fluorescent protein-Rev mutants indicated that basic residues within the KRRRK motif in the C-terminal region of Rev are necessary for targeting of Rev to the nucleus. Two separate regions of Rev were necessary for RNA binding: a central region encompassing residues 57 to 130 and a C-terminal region spanning residues 144 to 165. Within these regions were two distinct, short arginine-rich motifs essential for RNA binding, including an RRDRW motif in the central region and the KRRRK motif near the C terminus. These findings suggest that EIAV Rev utilizes a bipartite RNA-binding domain.     

Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6–8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6–8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation.     

Do viruses affect fecundity and survival of the copepod Acartia tonsa Dana?

Naturally occurring viruses are extremely abundant in aquaticsystems, and they infect bacteria, cyanobacteria, prokaryoticand eukaryotic phytoplankton, heterotrophic nanoflagellates,fish and mammals. Viral infections of single-celled organismshave been studied intensively in the past decade, but littleis known about the effects of viruses on aquatic metazoans,other than for some economically important species. Becausezooplankton assemblages are often dominated in number and biomassby copepods, we used them as model organisms to study the effectsof naturally occurring viruses on higher trophic levels. Weattempted to induce viral infection in laboratory-reared culturesof the estuarine copepod Acartia tonsa Dana by exposing themto elevated concentrations of natural viruses in seawater. Wefound no negative effects of such exposure on copepod fecundity,larval survival or adult survival.