Temporal kinetics and concentration-response relationships for induction of CYP1A, CYP2B, and CYP3A in primary cultures of beagle dog hepatocytes

Compared to other species, little information is available on the xenobiotic-induced regulation of cytochrome P450 enzymes in the beagle dog. Dogs are widely used in the pharmaceutical industry for many study types, including those that will impact decisions on compound progression. The purpose of this study was (1) to determine the temporal kinetics of drug-induced changes in canine CYP1A, CYP2B, and CYP3A mRNA and enzymatic activity, and (2) to characterize concentration-response relationships for CYP1A2, CYP2B11, and CYP3A12 using primary cultures of canine hepatocytes treated with beta-naphthoflavone (BNF), phenobarbital (PB), and rifampin (RIF), respectively. CYP1A1 and CYP1A2 mRNA exhibited maximal expression (12,700-fold and 206-fold, respectively) after 36 h of treatment with BNF. PB treatment, but not RIF treatment, caused maximal induction of CYP2B11 mRNA (149-fold) after 48 h of treatment. CYP3A12 and CYP3A26 mRNA levels were increased maximally after 72 h of treatment with PB and RIF (CYP3A12, 35-fold and 18-fold, and CYP3A26, 72-fold and 22-fold with PB and RIF treatment, respectively). Concentration-response relationships for BNF induced 7-ethoxyresorufin O-dealkylation (EROD) (EC(50) = 7.8 +/- 4.2 microM), PB induced 7-benzyloxyresorufin O-dealkylation (BROD) (EC(50) = 123 +/- 30 microM), and PB and RIF induced testosterone 6beta-hydroxylation (EC(50) = 132 +/- 28 microM and 0.98 +/- 0.16 microM) resembled the relationship for human CYP induction compared to that of rodent. Interestingly, RIF had no effect on CYP2B11 expression, which represents a species difference overlooked in previous investigations. Overall, the induction of dog CYP1A, CYP2B, and CYP3A exhibits characteristics that are intermediate to those of rodent and human.     

Stratification and intra- and inter-specific differences in fatty acid composition of common dolphin (Delphinus sp.) blubber: implications for dietary analysis

Sixty-five fatty acids were quantified in the blubber of common dolphins (Delphinus delphis, D. capensis) incidentally caught off the coast of southern California. Dolphins were grouped by sex, reproductive status and species, and a blubber sample was collected at a mid-lateral site located caudal to the trailing edge of the dorsal fin. Samples were divided horizontally into inner, middle and outer layers and gradients in fatty acid content (mass percent) were observed across the depth of the blubber. Levels of monounsaturated fatty acids were greatest in the outer layer, whereas levels of saturated and polyunsaturated fatty acids were greatest in the inner layer. Degree of stratification was greatest in sexually mature dolphins. Blubber of sexually immature, but physically mature, male dolphins was also highly stratified, suggesting that this difference may be attributed to differences in diet. Classification and regression tree analysis resulted in the fewest misclassifications when dolphins were grouped by species, possibly indicating that these closely related animals forage on different prey species. Dietary-derived fatty acids were typically selected as splitting criteria in classification and regression tree analyses, suggesting that the observed differences in fatty acid composition between the various groups of dolphins may be attributed to differences in diet.     

Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5

Background  

Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced.     

Fluorescent somatostatin receptor probes for the intraoperative detection of tumor tissue with long-wavelength visible light

Targeted fluorescent dyes are of substantial value for the intraoperative delineation of primary tumors and metastatic lesions. For this purpose long-wavelength red light (lambda=550-650 nm) offers advantages because of good tissue penetration and direct visibility. Since somatostatin receptors (SSTR) are overexpressed in a number of tumors, a series of potentially tumor-selective peptide-dye conjugates were synthesized by solid-phase peptide synthesis (SPPS). The octapeptides octreotate, Tyr(3)-octreotate and Tyr(3)-octreotide were employed and exhibited high affinity for somatostatin receptors (SSTR). The fluorescent dyes rhodamine 101, sulforhodamine B acid chloride, sulforhodamine 101 or rhodamine B isothiocyanate were conjugated either directly or via spacers, for example the peptidase-labile pentapeptide sequence Ala-Leu-Ala-Leu-Ala. The conjugates were completely assembled on the solid support: Fmoc-SPPS, cyclization via a disulfide linkage, N-terminal attachment of a spacer, and linkage to the fluorescent dye. An in vitro competition assay revealed that the conjugates bind to SSTRs with IC(50) values between 0.7 and 89 nM. The conjugates were generally stable to hydrolysis at pH 7-8 in buffer or serum. However, the rhodamine 101 conjugates revealed a loss of absorption at alkaline pH due to conversion to a neutral spirolactam form, as characterized by NMR.     

The synthesis and biological evaluation of a series of potent dual inhibitors of farnesyl and geranyl-Geranyl protein transferases

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.     

Detection and removal of contaminating hydroxylamines from the spin trap DEPMPO, and re-evaluation of its use to indicate nitrone radical cation formation and S(N)1 reactions

A previous report that the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) allows DEPMPO radical cation formation to be detected via the production of a carbon-centred radical adduct (assigned as the cis-hydroxyethyl species, formed by an intramolecular process) is shown to be incorrect. Rather, this and other paramagnetic species arise from the facile oxidation of trace hydroxylamine impurities present in commercial DEPMPO samples. As a result, techniques for the detection and elimination of such hydroxylamine impurities from DEPMPO solutions were developed and are described; these should prove to be of general use in EPR spin trapping experiments.     

Elevated serum silicon levels in women with silicone gel breast implants

The metatolic fate of silicone gel leaked from an intact or ruptured prosthesis is unknown. In this study, serum was blindly assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) for elemental silicon in 72 women with silicone gel breast implants and 55 control women (mean age 48 yr, both groups). Blood was drawn and processed using silicon-free materials. The mean silicon level in controls was 0.13±0.07 mg/L (range 0.06–0.35 mg/L), whereas in implant patients, the mean was significantly higher at 0.28±0.22 mg/L (range 0.06–0.87 mg/L) (P<0.01, Student'st-test with correction for unequal variances). Using the mean of the control group +2 SD as a cutoff for normal range (0.27 mg/L), 25/72 (34.7%) implant patients exceeded this value, compared with 2/55 (3.6%) controls. There was no significant correlation between past rupture of one or both implants, current rupture at the time of the blood draw, or the number of years with implants and silicon levels. The results suggest that serum silicon levels are elevated in many women with silicone gel breast implants. The chemical species involved and kinetics of this elevation remain to be determined.     

Spatial manipulation of cells and organelles using single electrode dielectrophoresis

The selection, isolation, and accurate positioning of single cells in three dimensions are increasingly desirable in many areas of cell biology and tissue engineering. We describe the application of a simple and low cost dielectrophoretic device for picking out and relocating single target cells. The device consists of a single metal electrode and an AC signal generator. It does not require microfabrication technologies or sophisticated electronics. The dielectrophoretic manipulator also discriminates between live and dead cells and is capable of redistributing intracellular organelles.     

Last of the human protists: the phylogeny and genetic diversity of Iodamoeba

Iodamoeba is the last genus of obligately parasitic human protist whose phylogenetic position is unknown. Iodamoeba small subunit ribosomal DNA sequences were obtained using samples from three host species, and phylogenetic analyses convincingly placed Iodamoeba as a sister taxon to Endolimax. This clade in turn branches among free-living amoeboflagellates of the genus Mastigamoeba. Two Iodamoeba ribosomal lineages (RL1 and RL2) were detected whose sequences differ by 31%, each of which is found in both human and nonhuman hosts.