A high-performance liquid chromatography/tandem mass spectrometric screening method for eight synthetic corticosteroids in bovine feces and the simultaneous differentiation between dexamethasone and betamethasone.

A screening method was developed to monitor the illegal use of synthetic corticosteroids in cattle. Diethyl ether extracts from spiked feces samples were cleaned-up by solid phase extraction followed by semipreparative reversed-phase chromatography (RPC). The fraction containing the corticosteroids was derivatized with ethoxyamine hydrochloride. The corresponding ethoximes were separated using silica-based C18 RPC and analyzed on-line in an ion trap mass spectrometer using atmospheric pressure positive chemical ionization. Ethoxime derivatives of dexamethasone and betamethasone were baseline resolved, allowing for the simultaneous mass spectrometric differentiation of both epimers in bovine feces by conventional non-chiral chromatography. At the lowest level tested (1 micro g/kg), corticosteroids (except triamcinolone) could be identified in compliance with the recent European criteria for residue identification. The quantitative performance of the method was best at residue levels > or = 2 micro g/kg.

Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein.

ALG-2 is a EF hand calcium binding protein with sequence homologies to calmodulin. Vito et al have shown that ALG-2 expression is required for apoptosis following a number of death stimuli,1 although nothing is known about the effectors which underlie ALG-2 function. Here we have used ALG-2 as bait in a yeast two hybrid screen of a mouse brain cDNA library. We found that ALG-2 binds to itself and to a novel protein that we call ALG-2 interacting protein X, Alix. Using co-immunoprecipitation experiments, we confirmed ALG-2/ALG-2 binding and demonstrated that this interaction is calcium independent. ALG-2/Alix interaction was also validated by co-immunoprecipitation, but in this case, the binding was found to be strictly calcium dependent. Alix seems highly conserved throughout evolution since it shows significant homologies to a putative C. elegans protein (YNK-1) and to proteins of A. nidulans (PalA) and S. cerevisiae (BRO1). Alix is a potential regulator or downstream effector of ALG-2 action.

Bax and Bak proteins require caspase activity to trigger apoptosis in sympathetic neurons.

We show that the pro-apoptotic proteins Bax and Bak trigger apoptosis when over-expressed in sympathetic neurons cultured in the presence of NGF. This effect can be blocked with z-VAD-fmk, a peptide inhibitor of caspases, but not with anti-apoptotic chemical compounds such as antioxidants or proteasome inhibitors. These results demonstrate that in sympathetic neurons Bax and Bak are sufficient to induce apoptosis in the absence of any other apparent cell death stimulus and that their effect is mediated by caspases but does not require reactive oxygen species nor activity of the proteasome.

Dissecting processing and apoptotic activity of a cysteine protease by mutant analysis.

We have compared the behavior of wild-type mouse NEDD-2, a neural precursor cell-expressed, developmentally down-regulated cysteine protease gene, to various mutant forms of the gene in both apoptotic activity in neuronal cells and proteolytic cleavage in the Semliki Forest virus and rabbit reticulocyte protein expression systems. Our results confirm that NEDD-2 processing and apoptotic activity are linked phenomena. They identify aspartate residues as likely targets for autocatalytic cleavage. They establish that cleavage events only occur at specific sites. Finally, they pinpoint differential effects of individual mutations on the overall proteolytic cleavage patterns, raising interesting questions related to the mechanisms of subunit assembly.

Function and expression of the Bcl-x gene in the developing and adult nervous system.

We have shown that overexpression of Bcl-x can rescue sympathetic neurones from nerve growth factor deprivation in vitro. We have also examined the distribution and expression of Bcl-x mRNA in the developing and adult nervous system using Northern blot and in situ hybridization. Bcl-x mRNA is widespread during development of the nervous system. In embryonic spinal cord, mRNA levels increase at the beginning of the naturally occurring cell death period, suggesting that Bcl-x may be involved in the selection of neurones during this period. In the brain, Bcl-x expression increases after birth to reach a high level in the adult brain. Neurones from the cortex, olfactory bulb, and Purkinje cells are among those expressing the highest levels of Bcl-x mRNA. The widespread expression of Bcl-x during development and in adult brain suggests of a role for Bcl-x beyond simply protecting neurones from developmental cell death.

Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation.

To study molecular mechanisms underlying neuronal cell death, we have used sympathetic neurons from superior cervical ganglia which undergo programmed cell death when deprived of nerve growth factor. These neurons have been microinjected with expression vectors containing cDNAs encoding selected proteins to test their regulatory influence over cell death. Using this procedure, we have shown previously that sympathetic neurons can be protected from NGF deprivation by the protooncogene Bcl-2. We now report that the E1B19K protein from adenovirus and the p35 protein from baculovirus also rescue neurons. Other adenoviral proteins, E1A and E1B55K, have no effect on neuronal survival. E1B55K, known to block apoptosis mediated by p53 in proliferative cells, failed to rescue sympathetic neurons suggesting that p53 is not involved in neuronal death induced by NGF deprivation. E1B19K and p35 were also coinjected with Bcl-Xs which blocks Bcl-2 function in lymphoid cells. Although Bcl-Xs blocked the ability of Bcl-2 to rescue neurons, it had no effect on survival that was dependent upon expression of E1B19K or p35.

Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia.

Naturally occurring cell death (NOCD) is a prominent feature of the developing nervous system. During this process, neurons express bcl-2, a major regulator of cell death whose expression may determine whether a neuron dies or survives. To gain insight into the possible role of bcl-2 during NOCD in vivo, we generated lines of transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase (NSE) or phosphoglycerate kinase (PGK) promoters. BCL-2 overexpression reduced neuronal loss during the NOCD period, which led to hypertrophy of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. Consistent with this finding, more axons than normal were found in the facial and optic nerves. We also tested whether neurons overexpressing BCL-2 were more resistant to permanent ischemia induced by middle cerebral artery occlusion; in transgenic mice, the volume of the brain infarction was reduced by 50% as compared with wild-type mice. These animals represent an invaluable tool for studying the effects of increased neuronal numbers on brain function as well as the mechanisms that control the survival of neurons during development and adulthood.

Metabolites of 4-chlorotestosterone acetate in cattle urine as diagnostic markers for its illegal use.

Seven metabolites of 4-chlorotestosterone acetate were identified in urine of cattle that received a single injection of the drug. The steroids were isolated by means of a series of clean-up steps carried out before and after enzymatic hydrolysis. The obtained extract was fractionated by high-performance liquid chromatography and each fraction was examined both by high-performance thin-layer chromatography and by capillary gas chromatography-mass spectrometry of the m-ethoxime-trimethylsilyl derivatives. The metabolites were tentatively identified by studying the mass spectra of selected peaks not found in blank samples. The structures of two metabolites, viz. 4-chloroandrost-4-ene-3,17-dione and 4-chloroandrost-4-ene-3 alpha,17 beta-diol were confirmed by chemical synthesis. The synthesized metabolites and 4-chloro-17 alpha-testosterone, a third metabolite which was identified tentatively, were located on the thin-layer chromatograms obtained. This study led to the conclusion that the illegal use of 4-chlorotestosterone acetate can be detected by identifying one or more of its metabolites in urine.

Molecular characterisation, expression and localisation of human neurokinin-3 receptor.

The complete amino acid sequence of the human neurokinin-3 receptor was deduced by DNA sequence analysis of human genomic fragments. Comparison of the predicted primary structure with those for the human neurokinin receptors 1 and 2 shows a highly conserved pattern of seven hydrophobic regions with maximum divergence occurring at the amino- and carboxy-termini. The position of intron-exon junctions are identical to those in other reported neurokinin genes. Using a chimeric genomic-cDNA gene, the human NK-3 receptor was expressed in Xenopus laevis oocytes where it mediates membrane conductance changes in response to its agonist, neurokinin B. More significantly, expression of the gene in mammalian cells resulted in detection of receptor binding as well as neurokinin-stimulated calcium mobilization and arachidonic acid release, all displaying the pharmacological characteristics expected of a neurokinin-3 receptor. By using the polymerase chain reaction we have shown that mRNA for the human neurokinin-3 receptor is expressed predominantly in the central nervous system.

Dexamethasone inhibits insulin biosynthesis by destabilizing insulin messenger ribonucleic acid in hamster insulinoma cells.

Glucocorticoid effects on insulin biosynthesis and secretion have been controversial. To define whether glucocorticoids affect insulin biosynthesis in vitro, the actions of dexamethasone on insulin gene expression were examined using the HIT cell line, a transformed clonal line of hamster beta-cells. Dexamethasone induced a dose-dependent decrease in steady-state insulin messenger RNA (mRNA) levels, which was prevented by adding an excess of RU 486, a competitive inhibitor for the binding to the glucocorticoid receptor. Inhibition was not observed before 6 h of dexamethasone treatment and was maximal at 24 h. To further assess the molecular mechanisms of the dexamethasone-induced decreases in insulin mRNA levels, we investigated whether transcription of the insulin gene was affected. Run-on assays revealed that transcription rates were not changed by glucocorticoids. Inhibition of RNA and protein synthesis by actinomycin D and cycloheximide, respectively, completely abolished the dexamethasone effects, whereas actinomycin D added 9 h after dexamethasone had no effect on insulin mRNA levels. The present results demonstrate that glucocorticoids can acutely inhibit insulin biosynthesis by destabilizing insulin mRNA; this effect requires the transcriptional activation of a gene encoding a protein responsible for the accelerated disappearance of insulin mRNA.

Functional characterization of a cAMP-responsive element of the rat insulin I gene.

Analogues of cAMP have been reported to increase insulin mRNA levels in normal rat beta-cells and hamster insulinoma cells (HIT). To define the mechanisms by which cAMP modulates insulin gene expression, we first investigated its effects on the transcriptional rate of the insulin gene in HIT cells. Nuclear run-on assays revealed a 4-fold increase in transcription observed as early as 1 h after stimulation. To characterize the cis-acting sequences of the rat insulin I gene promoter and the trans-acting factors mediating the cAMP effect on insulin gene transcription, we constructed DNA plasmids containing various lengths of the rat insulin I gene 5'-flanking region linked to the bacterial reporter gene, chloramphenicol acetyltransferase (CAT). Studies of the transcriptional activity of 5'-deletionally and pointly mutated plasmids after transfection into HIT cells revealed the presence of a cAMP-responsive element (CRE), TGACGTCC, between -177 and -184 relative to the transcriptional start site, whose sequence closely matches the previously defined CREs, present in cAMP-responsive genes. Gel retardation and Southwestern assays identify a protein of molecular weight approximately 43,000, binding specifically to the insulin CRE. We conclude that the rat insulin I gene is regulated by cAMP through a CRE and that the nuclear protein interacting with it might be similar or identical to the previously purified cAMP-responsive protein, CREB.