Effects of perinatal hypothyroidism on regulation of reelin and brain-derived neurotrophic factor gene expression in rat hippocampus: Role of DNA methylation and histone acetylation

Thyroid hormones have long been known to play important roles in the development and functions of the central nervous system, however, the precise molecular mechanisms that regulate thyroid hormone-responsive gene expression are not well understood. The present study investigated the role of DNA methylaion and histone acetylation in the effects of perinatal hypothyroidism on regulation of reelin and brain-derived neurotrophic factor (BDNF) gene expression in rat hippocampus. The findings indicated that the activities of DNA methyltransferase (DNMT), methylated reelin and BDNF genes were up-regulated, whereas, the activities of histone acetylases (HAT), the levels of global acetylated histone 3 (H3) and global acetylated histone 4 (H4), and acetylated H3, acetylated H4 at reelin promoter and at BDNF gene promoter for exon II were down-regulated in the hippocampus at the developmental stage of the hypothyroid animals. These results suggest that epigenetic modification of chromatin might underlie the mechanisms of hypothyroidism-induced down-regulation of reelin and BDNF gene expression in developmental rat hippocampus.     

Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato

A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4 degrees C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.     

Human anti-CXCR4 antibodies undergo VH replacement, exhibit functional V-region sulfation, and define CXCR4 antigenic heterogeneity

The chemokine receptor CXCR4 and its ligand stromal-derived factor-1 (SDF-1/CXCL12) are essential for many biological processes and various pathological conditions. However, the relationship between CXCR4 antigenic structure and SDF-1-mediated biological responses is poorly understood. In this report, a panel of human anti-CXCR4 Abs were isolated and used to explore CXCR4 antigenic heterogeneity and function. Multiple fixed CXCR4 antigenic isoforms were detected on the surface of hemopoietic cells. Epitope mapping studies demonstrated the complex nature of the surface-exposed CXCR4 epitopes. Ab-mediated inhibition of chemotaxis correlated strongly with binding affinity, epitope recognition, as well as the level of CXCR4 isoform expression. In addition, detailed genetic analyses of these Abs showed evidence of V(H) replacement. Importantly, structural and biochemical studies demonstrated tyrosine sulfation in novel regions of the V genes that contributed bidirectionally to the binding activity of the Abs. These data provide the first evidence that functional tyrosine sulfation occurs in self-reactive Abs and suggest a potential new mechanism that may contribute to the pathogenesis of Ab-mediated autoimmune disease. These Abs also provide valuable tools to explore the selective in vivo targeting of CXCR4 isoforms that may be preferentially expressed in certain disease states and involved in steady-state CXCR4-SDF-1 homeostasis.     

SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis

A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention.     

beta-Alkylthio indolyl carbinols: potent nonsteroidal antiandrogens with oral efficacy in a prostate cancer model

Through an in vivo screening model, we developed the in vivo SAR of beta-alkylthio indolyl carbinols. Through these efforts we identified a compound with potent oral in vivo efficacy in both immature and mature rat prostate weight reduction models and in a murine xenograft prostate cancer model.     

A selective androgen receptor modulator that reduces prostate tumor size and prevents orchidectomy-induced bone loss in rats

The pharmacological activity of JNJ-26146900 is described. JNJ-26146900 is a nonsteroidal androgen receptor (AR) ligand with tissue-selective activity in rats. The compound was evaluated in in vitro and in vivo models of AR activity. It binds to the rat AR with a K(i) of 400nM and acts as a pure androgen antagonist in an in vitro cell-based assay. Its in vitro profile is similar to the androgen antagonist bicalutamide (Casodex). In intact rats, JNJ-26146900 reduces ventral prostate weight with an oral potency (ED(50)) of 20-30mg/kg, again comparable to that of bicalutamide. JNJ-26146900 prevented prostate tumor growth in the Dunning rat model, maximally inhibiting growth at a dose of 10mg/kg. It slowed tumor growth significantly in a CWR22-LD1 mouse xenograft model of human prostate cancer. It was tested in aged male rats for its ability to prevent bone loss and loss of lean body mass following orchidectomy. After 6 weeks of dosing, bone volume decreased by 33% in orchidectomized versus intact vehicle-treated rats with a probability (P) of less than 0.05, as measured by micro-computerized tomography analysis. At a dose of 30mg/kg, JNJ-26146900 significantly reduced castration-induced tibial bone loss as indicated by the following parameters: bone volume, trabecular connectivity, trabecular number and spacing between trabeculae. Bone mineral density decreased from 229+/-34mg/cm(3) of hydroxyapatite to 166+/-26mg/cm(3) following orchidectomy, and was maintained at 194+/-20mg/cm(3) with JNJ-26146900 treatment (P<0.05 relative to orchidectomy alone). Using magnetic resonance imaging, the compound was found to partially prevent orchidectomy-induced loss of lean body mass. Our data show that selective androgen receptor modulators (SARMs) have the potential for anabolic effects on bone and muscle while maintaining therapeutic efficacy in prostate cancer.     

Parallel synthesis and SAR study of novel oxa-steroids as potent and selective progesterone receptor antagonists

Efficient parallel synthesis of novel 7-oxa-steroids 4 has been achieved from the key intermediate 3 via a one-pot four-step sequence. oxa-Steroids 4 with various ortho-, meta-, and para-monosubstituents on the phenyl ring, as well as disubstituted phenyl and heterocycles, were evaluated for progesterone receptor (PR) and glucocorticoid receptor (GR) antagonist activities. SAR study demonstrated that the para-fluorinated substituents on the phenyl ring not only increased the potency for PR in a T47D cell functional assay, but also improved the selectivity over GR in an A549 cell functional assay. The para-fluorophenyl oxa-steroid 4l and the para-trifluoromethylphenyl oxa-steroid 4p were found to be remarkably more potent and more selective PR antagonists than mifepristone, with subnanomolar potency and about 140-fold selectivity over GR. Molecular modeling of the oxa-steroid bound to PR provided meaningful insight for the SAR study. oxa-Steroids 4a and 4b were found to be more efficacious than mifepristone in vivo in a rat uterine complement C3 assay via the oral route, although they were less than or equally potent to mifepristone in the T47D assay.     

Synthesis and identification of novel oxa-steroids as progesterone receptor antagonists

A novel series of oxa-steroids 6 derived from (8S, 13S, 14R)-7-oxa-estra-4,9-diene-3,17-dione 1 have been synthesized and identified as potent and selective progesterone receptor antagonists. These novel oxa-steroids showed similar potency to mifepristone. Preliminary SAR study resulted in the most potent 17-phenylethynyl oxa-steroid 6i wih an IC(50) of 1.4nM. In contrast to the equipotent mifepristone toward the progesterone receptor (PR) and glucocorticoid receptor (GR), compound 6i had over 200-fold selectivity for PR over GR.     

Authentication of traditional Chinese medicine using infrared spectroscopy: distinguishing between ginseng and its morphological fakes

The quality of pharmaceutical products such as ginseng is important for ensuring consumer safety and efficacy. Ginseng is an expensive herb, and adulteration with other cheaper products may occur. Quality assurance of ginseng is needed since many of its commercial products now come in various formulations such as capsules, powder, softgels and tea. Thus traditional means of authentication via smell, taste or physical appearance are hardly reliable. Herbs like ginseng tend to exhibit characteristic infrared fingerprints due to their different chemical constituents. Here we report for the first time a rapid means of distinguishing American and Asian ginsengs from two morphological fakes – sawdust and Platycodon grandiflorum, via pattern differences and principal component analysis of their infrared spectra. Our results show that ginseng can be distinguished from both sawdust and Platycodon grandiflorum, hence there is a potential of using infrared spectroscopy as a novel analytical technique in the authentication of ginseng.     

iTRAQ-coupled 2D LC-MS/MS analysis on protein profile in vascular smooth muscle cells incubated with S- and R-enantiomers of propranolol: possible role of metabolic enzymes involved in cellular anabolism and antioxidant activity

Propranolol is a nonselective beta-blocker of the beta-adrenergic receptors, and the S-enantiomer is more active compared with the R-enantiomer. Clinically, it has been shown to be effective in hypermetabolic burn patients by decreasing cardiac work, protein catabolism, and lipolysis. While gene expression profiles have recently been reported in children receiving propranolol treatment, variations from one individual to another may have influenced the data analysis. Using iTRAQ-coupled 2D LC-MS/MS analysis, we report here the first study of protein profile in vascular smooth muscle cells incubated separately with the two enantiomers of propranolol. Four types of cellular proteins including metabolic enzymes, signaling molecules, cytoskeletal proteins, and those involved in DNA synthesis/protein translation displayed changes. The higher protein level of a number of enzymes involved in cellular anabolism and antioxidant activity in cells incubated with the S-enantiomer, as revealed by LC-MS/MS, was further supported by real-time PCR and Western blot analyses. Significantly, the increase in the anabolic activity associated with the higher level of metabolic enzymes was also supported by the higher intracellular concentration of the metabolic cofactor NAD+ which was a result of an increased oxidation of NADH. Our findings therefore provide molecular evidence on metabolic effect associated with propranolol treatment. The metabolic enzymes identified in our study may in turn be useful targets for future pharmaceutical interventions to reduce clinical side effects following propranolol treatment.