Assessment of developmental toxicity of vorinostat, a histone deacetylase inhibitor, in Sprague-Dawley rats and Dutch Belted rabbits

BACKGROUND: The developmental toxicity potential of vorinostat (suberoylanilide hydroxamic acid [SAHA], ZOLINZA), a potent inhibitor of histone deacetylase (HDAC), was assessed in Sprague-Dawley rats and Dutch Belted rabbits. HDAC inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation, and apoptosis of tumor cells. Range-finding studies established oral dose levels of 5, 15, or 50 mg/kg/day and 20, 50, or 150 mg/kg/day in rats and rabbits, respectively. METHODS: Animals were dosed on Gestation Days 6-20 or 7-20, respectively, with litter/fetal parameters evaluated on GD 21 and 28, respectively. Separate studies evaluated toxicokinetic parameters at the mid- and high-dose levels. RESULTS: There was no maternal toxicity observed at the highest dose levels; however, hematology and serum biochemistry changes were characterized in the range-finding studies. Vorinostat did not induce morphological malformations in either rat or rabbit fetuses. In rats, drug-related developmental toxicity was observed only in the high-dose group and consisted of markedly decreased fetal weight and increases in fetuses with a limited number of skeletal variations. In rabbits, drug-related developmental toxicity was also observed only in the high-dose group and consisted of slightly decreased fetal weight and increases in fetuses with a short 13th rib and incomplete ossification of metacarpals. Maternal exposures to vorinostat based on AUC and Cmax values were comparable at the high-dose levels of both species. Rabbits tolerated higher dosages probably due to more extensive metabolism. Maternal concentrations of vorinostat were approximately 1,000-fold above the known in vitro HDAC inhibitory concentration. CONCLUSIONS: Review of previous work with valproic acid, another HDAC inhibitor, suggest that the developmental toxicity profiles of these 2 compounds are not the result of HDAC inhibition but involve other mechanisms.     

An alternative Alcian Blue dye variant for the evaluation of fetal cartilage

BACKGROUND: The most comprehensive evaluation of vertebrate skeletal development involves the use of Alizarin Red S dye to stain ossified bone and various other dyes to stain cartilage. The dye used most widely to stain fetal cartilage in rodents and rabbits is Alcian Blue 8GX. However, the global supply of this specific dye has been exhausted. Several forms of the dye marketed as Alcian Blue 8GX are now available, although they are not synthesized via the original 8GX manufacturing process. METHODS: One new Alcian Blue 8GX form and two Alcian Blue dye variants were evaluated in rats and rabbits using standard staining procedures. The staining quality of these dyes were evaluated relative to the original form of Alcian Blue 8GX based on cartilage uptake of the dye, clarity of the cartilaginous components, staining intensity of the dye, and overall readability of the specimens under stereomicroscopic evaluation. RESULTS: Staining with the newer form of Alcian Blue 8GX resulted in poor staining quality. The Alcian Blue-Pyridine variant performed well, although staining intensity was less than optimal. The Alcian Blue-Tetrakis variant provided staining characteristics that were most similar to the original form of Alcian Blue 8GX. CONCLUSIONS: Alcian Blue-Tetrakis was markedly better in its ability to stain fetal cartilage than the newer form of Alcian Blue 8GX.     

Impact of human activities on the reproduction of Hooded Vultures Necrosyrtes monachus in Burkina Faso

During the last decades, the critically endangered Hooded Vulture Necrosyrtes monachus has strongly declined across its African range. Although direct persecution has been suggested as a major cause of this decline, little is known about the impact of humans on reproductive output in West Africa. We studied the impact of human activities on the reproductive output of Hooded Vultures in the Garango area of Burkina Faso. Twenty and 56 nesting attempts were monitored, respectively, during the breeding season in 2013/14 and 2014/15, to determine reproductive success and identify causes of nest failure. Annual breeding success varied between 0.68 and 0.71 chicks fledged per breeding pair per year and productivity was assessed at 0.57 chicks fledged per territorial pair in 2014/15. The main threats imposed by humans were poaching of eggs, chicks and collection of nest materials, leading to 20% (13 out of 64 breeding attempts) of nest failures over the two years. An additional important reason for nest failure was the pruning and (partial) cutting of nest trees. Despite this high level of human interference, we found that Hooded Vulture nest success increased with proximity to human settlements, probably because breeding vultures benefit from protection by people against persecution and disturbance.     

Functional analysis of a human A(1) adenosine receptor/green fluorescent protein/G(i1)alpha fusion protein following stable expression in CHO cells

The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease.     

GABA(B) receptors function as heterodimers

Our current understanding is that functional GABA(B) receptors exist as heterodimers of two related seven-transmembrane proteins, GABA(B)-R1 and GABA(B)-R2. GABA(B)-R1 requires GABA(B)-R2 to be expressed at the cell surface as a mature glycoprotein. Cloning of the GABA(B) receptor has failed to provide molecular evidence to support the existence of true receptor subtypes. The discovery of the heterodimeric nature of the GABA(B) receptor has already changed the way we think about GPCR function and it is likely that future studies will change our understanding about how receptor subtypes can be formed.     

Temporal integration in nasal lateralization and nasal detection of carbon dioxide

Two experiments examined time/concentration trading for the detection of carbon dioxide, an irritant with little or no odor. Experiment 1 employed the nasal lateralization method: subjects attempted to determine which nostril received carbon dioxide and which received pure air when presented simultaneously. Experiment 2 employed a temporal, two-alternative, forced-choice, detection paradigm with monorhinal stimulation. In both experiments, stimulus duration was varied at a number of fixed concentrations to determine the shortest, detectable pulse. Under both conditions, threshold pulse duration decreased as stimulus concentration increased. Power functions with exponents of less than negative one described the data quite well: More than a twofold increase in duration was needed to compensate for a twofold decrease in concentration. Thus, for carbon dioxide, the nasal trigeminal system functions as an imperfect integrator at threshold-level.     

Confirmation of data mining based predictions of protein function

MOTIVATION: A central problem in bioinformatics is the assignment of function to sequenced open reading frames (ORFs). The most common approach is based on inferred homology using a statistically based sequence similarity (SIM) method, e.g. PSI-BLAST. Alternative non-SIM based bioinformatic methods are becoming popular. One such method is Data Mining Prediction (DMP). This is based on combining evidence from amino-acid attributes, predicted structure and phylogenic patterns; and uses a combination of Inductive Logic Programming data mining, and decision trees to produce prediction rules for functional class. DMP predictions are more general than is possible using homology. In 2000/1, DMP was used to make public predictions of the function of 1309 Escherichia coli ORFs. Since then biological knowledge has advanced allowing us to test our predictions. RESULTS: We examined the updated (20.02.02) Riley group genome annotation, and examined the scientific literature for direct experimental derivations of ORF function. Both tests confirmed the DMP predictions. Accuracy varied between rules, and with the detail of prediction, but they were generally significantly better than random. For voting rules, accuracies of 75-100% were obtained. Twenty-one of these DMP predictions have been confirmed by direct experimentation. The DMP rules also have interesting biological explanations. DMP is, to the best of our knowledge, the first non-SIM based prediction method to have been tested directly on new data. AVAILABILITY: We have designed the "Genepredictions" database for protein functional predictions. This is intended to act as an open repository for predictions for any organism and can be accessed at http://www.genepredictions.org