Protein expression profiling of the drosophila fragile X mutant brain reveals up-regulation of monoamine synthesis

Fragile X syndrome is the most common form of inherited mental retardation, associated with both cognitive and behavioral anomalies. The disease is caused by silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the mRNA-binding, translational regulator FMRP. Previously we established a disease model through mutation of Drosophila fmr1 (dfmr1) and showed that loss of dFMRP causes defects in neuronal structure, function, and behavioral output similar to the human disease state. To uncover molecular targets of dFMRP in the brain, we use here a proteomic approach involving two-dimensional difference gel electrophoresis analyses followed by mass spectrometry identification of proteins with significantly altered expression in dfmr1 null mutants. We then focus on two misregulated enzymes, phenylalanine hydroxylase (Henna) and GTP cyclohydrolase (Punch), both of which mediate in concert the synthetic pathways of two key monoamine neuromodulators, dopamine and serotonin. Brain enzymatic assays show a nearly 2-fold elevation of Punch activity in dfmr1 null mutants. Consistently brain neurochemical assays show that both dopamine and serotonin are significantly increased in dfmr1 null mutants. At a cellular level, dfmr1 null mutant neurons display a highly significant elevation of the dense core vesicles that package these monoamine neuromodulators for secretion. Taken together, these data indicate that dFMRP normally down-regulates the monoamine pathway, which is consequently up-regulated in the mutant condition. Elevated brain levels of dopamine and serotonin provide a plausible mechanistic explanation for aspects of cognitive and behavioral deficits in human patients.     

Use of pressurized liquid extraction (PLE)/gas chromatography-electron capture detection (GC-ECD) for the determination of biodegradation intermediates of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in soils

A rapid, sensitive, and reproducible method was developed for quantitative determination of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and its biodegradation intermediates, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) in soils. RDX, MNX, DNX, or TNX was extracted from soil by pressurized liquid extraction (PLE), followed by cleanup using florisil. Instrumental analysis was performed using gas chromatography with electron capture detection (GC-ECD), which was highly sensitive to the parent explosive and its metabolites. The method detection limits (MDLs) were 0.243, 0.095, 0.138, and 0.057 ng/g for RDX, MNX, DNX, and TNX, respectively. The method gave high recovery (98-102%), good precision (0.22-5.14%), and reproducibility, and proved to be suitable for real world sample analysis.     

Capillary electrophoresis analysis of mangiferin extracted from Mangifera indica L. bark and Mangifera persiciformis C.Y. Wu et T.L. Ming leaves

The flavonoid compound mangiferin is found in the leaves, stem bark, fruit peels and root of Mangifera indica L. and in many other herbal species with many potential pharmacological properties. We have established an analytical method of mangiferin extracted from M. indica L. bark and Mangifera persiciformis C.Y. Wu et T.L. Ming leaves utilizing CZE. An electrolytic buffer containing 0.05 M borate buffer, pH 7.4 with methanol (1:0.3, v/v) was deemed suitable for mangiferin analysis. An ideal mangiferin electropherogram with a migration time at approximately 10.50 min was obtained. Repeatability tests showed that the R.S.D.s for both intra- and inter-day migration time and peak area for all manigferin sources tested were less than 4%. The linearity range of this method was 5-1000 microg/ml. The detection limit of this method was 1.5 microg/ml. Quantitative analysis of mangiferin was also performed with this method. The accuracy of quantitation at 10, 500 and 1000 microg/ml of control mangiferin were 99.00, 99.38 and 99.14%, respectively (n=10). The repeatability of quantitation (R.S.D.) was below 3%. Our results demonstrated that CZE is a simple and reliable method in mangiferin analysis and more studies are needed to detect other mangiferin resources, such as clinical biological samples, in pharmacology and pharmacokinetic studies.     

Cytotoxic activity and constituents of the volatile oil from the roots of Patrinia scabra Bunge

The volatile oil from the roots of Patrinia scabra Bunge was isolated by steam distillation, and separated into four major fractions (Fr. A-D) by means of column chromatography. A total of 39 compounds (1-39) were identified by GC/MS analysis, and evaluated for their in vitro cytotoxic activities against human ovarian carcinoma cells (HO-8910) and human hepatoma cells (Bel-7402) (Table 1). Fr. A showed the strongest inhibitory effect on HO-8910 (IC50 = 21 microg/ml) and Bel-7402 cells (16 mcirog/ml), whereas Fr. B was the least active (>100 microg/ml). By comparison of the constituents of the four fractions, we assume that the cytotoxicity of the volatile oil of P. scabra is mainly due to the lignans and azulenes, rather than to caryophyllene oxide I (18). Our results suggest that the volatile oil of P. scabra possesses potent and tumor-specific cytotoxicity, and could serve as a possible candidate for future cancer chemotherapy.     

Biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and 229E human coronaviruses

Severe acute respiratory syndrome (SARS) is a serious health threat and its early diagnosis is important for infection control and potential treatment of the disease. Diagnostic tools require rapid and accurate methods, of which a capture ELISA method may be useful. Toward this goal, we have prepared and characterized soluble full-length nucleocapsid proteins (N protein) from SARS and 229E human coronaviruses. N proteins form oligomers, mostly as dimers at low concentration. These two N proteins degrade rapidly upon storage and the major degraded N protein is the C-terminal fragment of amino acid (aa) 169-422. Taken together with other data, we suggest that N protein is a two-domain protein, with the N-terminal aa 50-150 as the RNA-binding domain and the C-terminal aa 169-422 as the dimerization domain. Polyclonal antibodies against the SARS N protein have been produced and the strong binding sites of the anti-nucleocapsid protein (NP) antibodies produced were mapped to aa 1-20, aa 150-170 and aa 390-410. These sites are generally consistent with those mapped by sera obtained from SARS patients. The SARS anti-NP antibody was able to clearly detect SARS virus grown in Vero E6 cells and did not cross-react with the NP from the human coronavirus 229E. We have predicted several antigenic sites (15-20 amino acids) of S, M and N proteins and produced antibodies against those peptides, some of which could be recognized by sera obtained from SARS patients. Antibodies against the NP peptides could detect the cognate N protein clearly. Further refinement of these antibodies, particularly large-scale production of monoclonal antibodies, could lead to the development of useful diagnostic kits for diseases associated with SARS and other human coronaviruses.     

Immunological-adjuvant saponins from the roots of Panax notoginseng

The total saponin extract from the dried roots of Panax notoginseng (Burk.) F. H. Chen possesses immunological-adjuvant activities. Guided by in vivo immunological tests, further study on this fraction afforded three active dammarane-type saponins. Their structures were determined on the basis of chemical evidence and extensive spectroscopic methods, including 1D- and 2D-NMR. The novel compound (20S)-protopanaxatriol 20-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (1), and the two known compounds ginsenoside Rh4 (2) and notoginsenoside K (3) exhibited immunological-adjuvant activities on the humoral immune responses of ICR mice against ovalbumin (OVA).     

Chromatin modification and the endothelial-specific activation of the E-selectin gene

E-selectin plays a role in the binding and extravasation of leukocytes from the bloodstream. The E-selectin gene is rapidly and transiently expressed by endothelial cells activated by inflammatory stimuli. Despite the identification of factors critical for cytokine-induced activation of the E-selectin promoter, little is known about the mechanisms that restrict the gene expression to endothelial cells. We used in vivo approaches to characterize the E-selectin promoter in primary cultures of human umbilical vein endothelial cells and umbilical artery smooth muscle cells. In endothelial cells specifically, nucleosomes are remodeled after tumor necrosis factor (TNF) alpha induction. Chromatin immunoprecipitation analysis demonstrated the binding of the p65 (RelA) component of nuclear factor-kappa B (NF-kappa B) to the endogenous E-selectin promoter after TNFalpha stimulation along with IkappaB kinase alpha. Multiple coactivators, including p300, steroid receptor coactivator-1, and p300/cAMP-response element-binding protein (CREB)-binding protein (CBP)-associated factor localize differentially to the E-selectin promoter. Additionally, TNFalpha induced localized histone hyperacetylation, phosphorylation, and methylation in the E-selectin gene specifically in endothelial cells. Post-induction repression of E-selectin expression is associated with recruitment of multiple deacetylases. Collectively, these studies suggest a model for the selective induction of the E-selectin gene in which the core promoter chromatin architecture is specifically modified in endothelial cells.     

Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site

Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/c)(A/t)A*GNNCt(T/a)N(C/a) and GN4G(G/c)(A/c)G*GNNCtTN(C/a) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between -1 and +1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. +1G/+4C, -4G/+8C, and particularly the novel -2A/+6T, but with no preference at +2/+3 within the staggered 4-bp overhang. Asymmetric elements include -3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique -2A/+6T and other preferences.