Increased expression of acyl-coenzyme A: cholesterol acyltransferase-1 and elevated cholesteryl esters in the hippocampus after excitotoxic injury.

Significant increases in levels of cholesterol and cholesterol oxidation products are detected in the hippocampus undergoing degeneration after excitotoxicity induced by the potent glutamate analog, kainate (KA), but until now, it is unclear whether the cholesterol is in the free or esterified form. The present study was carried out to examine the expression of the enzyme involved in cholesteryl ester biosynthesis, acyl-coenzyme A: cholesterol acyltransferase (ACAT) and cholesteryl esters after KA excitotoxicity. A 1000-fold greater basal mRNA level of ACAT1 than ACAT2 was detected in the normal brain. ACAT1 mRNA and protein were upregulated in the hippocampus at 1 and 2 weeks after KA injections, at a time of glial reaction. Immunohistochemistry showed ACAT1 labeling of oligodendrocytes in the white matter and axon terminals in hippocampal CA fields of normal rats, and loss of staining in neurons but increased immunoreactivity of oligodendrocytes, in areas affected by KA. Gas chromatography-mass spectrometry analyses confirmed previous observations of a marked increase in level of total cholesterol and cholesterol oxidation products, whilst nuclear magnetic resonance spectroscopy showed significant increases in cholesteryl ester species in the degenerating hippocampus. Upregulation of ACAT1 expression was detected in OLN93 oligodendrocytes after KA treatment, and increased expression was prevented by an antioxidant or free radical scavenger in vitro. This suggests that ACAT1 expression may be induced by oxidative stress. Together, our results show elevated ACAT1 expression and increased cholesteryl esters after KA excitotoxicity. Further studies are necessary to determine a possible role of ACAT1 in acute and chronic neurodegenerative diseases.

The rapid identification of Clostridium perfringens as the possible aetiology of a diarrhoeal outbreak using PCR.

A gastroenteritis outbreak occurred in a military camp where a laboratory and epidemiological investigation was carried out. The early onset of symptoms indicated probable food contamination with Clostridium perfringens. Stool samples collected from affected patients were tested within 4 h via real-time polymerase chain reaction (PCR) for the presence of the C. perfringens plc gene. Ten out of the 12 stool samples were positive. Confirmation of the molecular test results was carried out by enumeration of C. perfringens in stool by culture and shown to be in excess of 106 spores/g stool. The isolates obtained from culture were further analysed by PCR for the presence of the chromosomal enterotoxin (cpe) gene. Based on the clinical symptoms, epidemiological and laboratory investigations, C. perfringens was implicated as the aetiological agent. The ability to conduct real-time PCR analysis greatly shortens the time to diagnosis and allows for preventive and control measures to be effected quickly.

Determination of berberine and strychnine in medicinal plants and herbal preparations by pressurized liquid extraction with capillary zone electrophoresis.

A simple and rapid method for the determination of berberine and strychnine in medicinal plants and herbal preparations for regulatory purposes using a home-made pressurized liquid extraction (PLE) system with capillary zone electrophoresis (CZE) using ultraviolet detection at 254 nm was developed. The effects of pH, concentration of buffer, and organic modifiers in the electrophoretic separation were investigated. The buffer used for CZE contained 50 mM ammonium acetate, pH 3.1. The effect of temperature on the extraction efficiency of strychnine in medicinal plants by PLE was demonstrated. Comparable or higher extraction efficiency was achieved with PLE for strychnine in medicinal plants and berberine in herbal preparations compared to soxhlet extraction. The effect of matrix interference in medicinal plants and herbal preparations containing a number of medicinal plants samples using CZE was investigated by standard additional experiments. The reproducibility of the method using PLE with CZE was found to vary between 2.4 and 10.7% (n = 5/6) for different types of samples on different days.

Determination of aristolochic acids in medicinal plants (Chinese) prepared medicine using capillary zone electrophoresis.

Aristolochic acids (I and II) are commonly found in medicinal plants such as Radix aristolochiae and have been reported to cause acute hepatitis and end-stage renal failure. The aim of this work was to develop a method for the analysis of aristolochic acids in medicinal plant/Chinese prepared medicine (CPM) using (CZE). The buffer used was 30 mM sodium tetraborate at pH 9.5, detection was at 254 nm, applied voltage at 18 kV and the temperature was set at 25 degrees C. The effect of ionic strength, pH, and applied voltage on the separation was investigated. The precision values (relative standard deviation, RSD, %) for the relative migration time and peak area or peak height for aristolochic acids I and II were found to be less than 0.3% and between 2.6 to 4.0%, respectively. The limit of detection for aristolochic acids I and II was found to be 1.2 and 0.9 mg/L, respectively. The proposed method using pressurized liquid extraction (PLE) with CZE was used to determine the amount of aristolochic acids in medicinal plants or CPM samples with complex matrix and the results were compared with high-performance liquid chromatography (HPLC). Method precision (RSD, n = 6) was found to be less than 4% when those from applied to medicinal plants and CPM samples.

An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids.

Hepatic hydroxylation is an essential step in the metabolism and excretion of bile acids and is necessary to avoid pathologic conditions such as cholestasis and liver damage. In this report, we demonstrate that the human xenobiotic receptor SXR (steroid and xenobiotic receptor) and its rodent homolog PXR (pregnane X receptor) serve as functional bile acid receptors in both cultured cells and animals. In particular, the secondary bile acid derivative lithocholic acid (LCA) is highly hepatotoxic and, as we show here, a metabolic substrate for CYP3A hydroxylation. By using combinations of knockout and transgenic animals, we show that activation of SXR/PXR is necessary and sufficient to both induce CYP3A enzymes and confer resistance to toxicity by LCA, as well as other xenotoxicants such as tribromoethanol and zoxazolamine. Therefore, we establish SXR and PXR as bile acid receptors and a role for the xenobiotic response in the detoxification of bile acids.

Determination of lead in botanicals/Chinese prepared medicines by using microwave digestion with flow injection-inductively coupled plasma-mass spectrometry.

A simple, sensitive, and rapid method was developed for the determination of lead in botanicals and Chinese prepared medicines (CPM) by using closed-vessel microwave digestion with flow injection-inductively coupled plasma-mass spectrometry. The limits of detection and quantitation for the method, based on 0.5 g digested sample, were calculated to be 0.10 and 0.61 mg/kg, respectively. A simple approach was proposed for calibration by multiple linear regression (MLR) with 207Pb, 206Pb, and 204Pb for the determination of lead in botanical and CPM samples. The results from calibration by MLR were compared with those obtained by conventional modes using 207Pb and the sum of 207Pb and 206Pb. The results obtained by the different modes of calibration were in good agreement for botanical and CPM samples. The method was found to have good accuracy for the analysis of botanical reference materials. Method precision based on analyses of different types of CPM samples by different analysts on different days for different levels of lead was between 3.0 and 8.0% (relative standard deviation, n = 6). The effect of possible matrix interference caused by nitric acid and the extent of digestion was investigated with the method of standard additions. Significant matrix interference was not observed for the CPM samples analyzed. MLR was used to examine the effect of variation in isotopic abundance, which was found to present no significant problem in the determination of lead in the botanical and CPM samples.

Pressurized liquid extraction of berberine and aristolochic acids in medicinal plants.

Berberine and aristolochic acids I and II present naturally in medicinal plants were extracted using a laboratory-made pressurized liquid extraction (PLE) system in the dynamic mode. As the target analytes were present naturally in the medicinal plants, spiking was not done and comparison with ultrasonic extraction and Soxhlet extraction was performed to assess the method accuracy. The effect of temperature, volume of solvent required and particle size were investigated. Method precision (RSD, n=5) between 1.98 and 3.4% was achieved for the extraction of berberine and aristolochic acids I and II in medicinal plants and lower than 8% for lower levels of aristolochic acid II in medicinal plants.

PPAR gamma is required for placental, cardiac, and adipose tissue development.

The nuclear hormone receptor PPAR gamma promotes adipogenesis and macrophage differentiation and is a primary pharmacological target in the treatment of type II diabetes. Here, we show that PPAR gamma gene knockout results in two independent lethal phases. Initially, PPAR gamma deficiency interferes with terminal differentiation of the trophoblast and placental vascularization, leading to severe myocardial thinning and death by E10.0. Supplementing PPAR gamma null embryos with wild-type placentas via aggregation with tetraploid embryos corrects the cardiac defect, implicating a previously unrecognized dependence of the developing heart on a functional placenta. A tetraploid-rescued mutant surviving to term exhibited another lethal combination of pathologies, including lipodystrophy and multiple hemorrhages. These findings both confirm and expand the current known spectrum of physiological functions regulated by PPAR gamma.

Determination of arsenic in traditional Chinese medicine by microwave digestion with flow injection-inductively coupled plasma mass spectrometry (FI-ICP-MS).

A simple, rapid, and sensitive method with high sample throughput was developed for determining arsenic in traditional Chinese medicine (TCM) in the form of uncoated tablets, sugar-coated tablets, black pills, capsules, powders, and syrups. The method involves microwave digestion with flow injection-inductively coupled plasma mass spectrometry (FI-ICP-MS). Method precision was 2.7-10.1% (relative standard deviation, n = 6) for different concentrations of arsenic in different TCM samples analyzed by different analysts on different days. Method accuracy was checked with a certified reference material (sea lettuce, Ulva lactuca, BCR CRM 279) for external calibration and by spiking arsenic standard into different TCMs. Recoveries of 89-92% were obtained for the certified reference material and higher than 95% for spiked TCMs. Matrix interference was insignificant for samples analyzed by the method of standard addition. Hence, no correction equation was used in the analysis of arsenic in the samples studied. Sample preparation using microwave digestion gave results that were very similar to those obtained by conventional wet acid digestion using nitric acid.

SXR, a novel steroid and xenobiotic-sensing nuclear receptor.

An important requirement for physiologic homeostasis is the detoxification and removal of endogenous hormones and xenobiotic compounds with biological activity. Much of the detoxification is performed by cytochrome P-450 enzymes, many of which have broad substrate specificity and are inducible by hundreds of different compounds, including steroids. The ingestion of dietary steroids and lipids induces the same enzymes; therefore, they would appear to be integrated into a coordinated metabolic pathway. Instead of possessing hundreds of receptors, one for each inducing compound, we propose the existence of a few broad specificity, low-affinity sensing receptors that would monitor aggregate levels of inducers to trigger production of metabolizing enzymes. In support of this model, we have isolated a novel nuclear receptor, termed the steroid and xenobiotic receptor (SXR), which activates transcription in response to a diversity of natural and synthetic compounds. SXR forms a heterodimer with RXR that can bind to and induce transcription from response elements present in steroid-inducible cytochrome P-450 genes and is expressed in tissues in which these catabolic enzymes are expressed. These results strongly support the steroid sensor hypothesis and suggest that broad specificity sensing receptors may represent a novel branch of the nuclear receptor superfamily.

LXR, a nuclear receptor that defines a distinct retinoid response pathway.

We have identified a new retinoid response pathway through which 9-cis retinoic acid (9cRA) activates transcription in the presence of LXR alpha, a member of the nuclear receptor superfamily. LXR alpha shows a specific pattern of expression in visceral organs, thereby restricting the response to certain tissues. Retinoid trans-activation occurs selectively on a distinct response element termed an LXRE. Significantly, neither RXR homodimers nor RXR/RAR heterodimers are able to substitute for LXR alpha in mediating this retinoid response. We provide evidence that the retinoid response on the LXRE is the result of a unique interaction between LXR alpha and endogenous RXR, which, unlike in the RXR/RAR heterodimer, makes RXR competent to respond to retinoids. Thus, the interaction with LXR alpha shifts RXR from its role described previously as a silent, DNA-binding partner to an active ligand-binding subunit in mediating retinoid responses through target genes defined by LXREs.

Cross-talk among ROR alpha 1 and the Rev-erb family of orphan nuclear receptors.

We have cloned Rev-erb beta, a novel isoform of the Rev-erb alpha orphan nuclear receptor. The DNA binding domains of Rev-erb alpha and beta are highly related to each other and to the retinoic acid related orphan receptor (ROR)/RZR subfamily of nuclear receptors. Indeed, we find that all three receptors bind as monomers to the sequence AATGT-AGGTCA. Whereas ROR alpha 1 constitutively activates transcription through this sequence, both isoforms of Rev-erb are inactive. When coexpressed, both Rev-erb isoforms suppress the transcriptional activity of ROR alpha 1. Our data define Rev-erb and ROR/RZR as a family of related receptors with opposing activities on overlapping regulatory networks.

Differential expression and activation of a family of murine peroxisome proliferator-activated receptors.

To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in mammals, we have cloned and characterized two PPAR alpha-related cDNAs (designated PPAR gamma and -delta, respectively) from mouse. The three PPAR isoforms display widely divergent patterns of expression during embryogenesis and in the adult. Surprisingly, PPAR gamma and -delta are not activated by pirinixic acid (Wy 14,643), a potent peroxisome proliferator and activator of PPAR alpha. However, PPAR gamma and -delta are activated by the structurally distinct peroxisome proliferator LY-171883 and linoleic acid, respectively, indicating that each of the isoforms can act as a regulated activator of transcription. These data suggest that tissue-specific responsiveness to peroxisome proliferators, including certain fatty acids, is in part a consequence of differential expression of multiple, pharmacologically distinct PPAR isoforms.

Chromosomal localization of the human retinoid X receptors.

The recently described retinoid X receptors (RXRs) respond to the novel retinoid 9-cis-retinoic acid and also serve as heterodimeric partners for the vitamin D, thyroid hormone, and retinoic acid receptors (VDR, TR, and RAR, respectively). In this work, we report high-resolution localization of the human RXR genes within cytogenetic bands and also within a standard reference map of cosmid DNA markers on human chromosomes. We have determined the location of the human RXR genes by pairwise hybridization of the RXR cosmids and reference markers, using fluorescence in situ hybridization. We localized (i) RXR alpha (RXRA) to chromosome 9 band q34.3; (ii) RXR beta (RXRB) to chromosome 6 band 21.3; and (iii) RXR gamma (RXRG) to chromosome 1 band q22-q23. Six retinoid-responsive transcription factors have been identified so far, including three retinoic acid receptors in addition to the three RXRs. Interestingly, each of these receptors in human and mouse is encoded by genes located at distinct chromosomal loci and on separate chromosomes. The proximity of RXR genes to loci known to be associated with genetic disorders suggests that their location may be useful in establishing a link between RXRs and certain human diseases.

Characterization of three RXR genes that mediate the action of 9-cis retinoic acid.

An understanding of the differences and similarities of the retinoid X receptor (RXR) and retinoic acid receptor (RAR) systems requires knowledge of the diversity of their family members, their patterns of expression, and their pharmacological response to ligands. In this paper we report the isolation of a family of mouse RXR genes encoding three distinct receptors (RXR alpha, beta, and gamma). They are closely related to each other in their DNA- and ligand-binding domains but are quite divergent from the RAR subfamily in both structure and ligand specificity. Recently, we demonstrated that all-trans retinoic acid (RA) serves as a "pro-hormone" to the isomer 9-cis RA, which is a high-affinity ligand for the human RXR alpha. We extend those findings to show that 9-cis RA is also "retinoid X" for mouse RXR alpha, beta, and gamma. Trans-activation analyses show that although all three RXRs respond to a variety of endogenous retinoids, 9-cis RA is their most potent ligand and is up to 40-fold more active than all-trans RA. Northern blot and in situ hybridization analyses define a broad spectrum of expression for the RXRs, which display unique patterns and only partially overlap themselves and the RARs. This study suggests that the RXR family plays critical roles in diverse aspects of development, from embryo implantation to organogenesis and central nervous system differentiation, as well as in adult physiology.

The occurrence of periodontitis-related microorganisms in relation to titanium implants.

The peri-implant space was examined for the presence of periodontitis-related microorganisms in 19 patients. All patients had been fitted with osseointegrated titanium implants which were clinically sound at the time of examination and all had been prescribed daily 0.2% chlorhexidine mouthrinse. In 17 patients, two contralateral fixtures in patients were selected, the remaining 2 patients had only one fixture, and associated mucosal clinical variables assessed for plaque accumulation, gingival inflammation, bleeding tendency, and probing depth. The bacteriological composition of submucosal plaque samples was investigated using non-selective media and selective media for Actinobacillus actimomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Prevotella intermedia (Pi). The peri-implant tissues were healthy. Twenty-two of 37 sites had a greater proportion of anaerobes than aerobes. Aa was detected at one site. Pg was not cultivated, and Pi was present in 7 of 37 sites, comprising between 0.4% and 60.9% of the total anaerobic viable counts. Since the presence of any of these organisms may play a contributory role to the loss of the fixture, it is recommended that the submucosal plaque of implants be monitored regularly for the presence of these periodontitis-associated species.

A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR.

The vitamin A derivative retinoic acid exerts its effects on transcription through two distinct classes of nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR). We provide evidence that expression of the gene for cellular retinol-binding protein type II (CRBPII), a key protein in the intestinal absorption of vitamin A, is dramatically up-regulated by retinoic acid in the presence of RXR but not RAR. This regulation is conferred through a specific cis element in the CRBPII promoter that contains five nearly perfect tandem repeats of the sequence AGGTCA spaced by a single nucleotide. The discovery of this new RX response element provides a means for distinguishing between the two retinoid receptor systems and suggests that an RXR-mediated pathway exists for modulating vitamin A metabolism.

Nuclear receptor that identifies a novel retinoic acid response pathway.

Molecular cloning and transcriptional activation studies have revealed a new protein similar to the steroid hormone receptors and which responds specifically to vitamin A metabolites. This protein is substantially different in primary structure and ligand specificity from the products of the previously described retinoic acid receptor gene family. By indicating the existence of an additional pathway through which retinoic acid may exert its effects, these data lead to a re-evaluation of retinoid physiology.