CTLA4 Autoimmunity-Associated Genotype Contributes to Severe Pulmonary Tuberculosis in an African Population

The gene of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA4), a negative regulator of T lymphocytes, contains a single-nucleotide polymorphism (SNP) at position +6230A->G (ct60A->G), which has been found associated with several autoimmune diseases and appears to reduce T-cell inhibitory activity. In Ghana, West Africa, we compared the frequencies of CTLA4 +6230 A/G and 6 haplotype-tagging SNPs in 2010 smear-positive, HIV-negative patients with pulmonary tuberculosis (TB) and 2346 controls matched for age, gender and ethnicity. We found no difference in allele frequencies between cases and controls. However, +6230A and a distinct CTLA4 haplotype and a diplotype comprising the +6230A allele were significantly less frequent among cases with large opacities in chest radiographs compared to those with small ones (P_{corrected [cor]} = 0.002, P_cor = 0.00045, P = 0.0005, respectively). This finding suggests that an increased T-cell activity associated with the CTLA4 +6230G allele contributes to pathology rather than to protection in pulmonary TB.     

Glucoraphasatin: Chemistry, occurrence, and biological properties

Glucoraphasatin is an atypical glucosinolate mainly found in Raphanus sativus roots and sprouts. This review focuses on the chemistry, the occurrence, and the biological properties of glucoraphasatin.     

In situ nick translation of metaphase chromosomes with biotin-labeled d-UTP

Summary Conditioned culture medium from Daudi cells was used as a source of soluble H-Y antigen. Concentrated culture medium was labeled with ¹²⁵I and then fractionated by gel filtration. Column fractions were assayed for the presence of H-Y antigen by urease-ELISA. H-Y antigen-containing fractions were then pooled and subjected to an improved immunoprecipitation protocol. Three predominant H-Y antigenic proteins were identified with estimated molecular weights of above 200,000, 50,000, and 20,000.     

UDP-N-acetylglucosamine 1-carboxyvinyl-transferase from Acinetobacter calcoaceticus

Abstract We have analyzed the sequence downstream of rpoN from Zcinetobacter calcoaceticus and identified an open reading frame encoding a protein with high similarity to UDP- N -acetylgucosamine 1-carboxyvinyl-transferase (MurZ). Multicopy plasmids encoding this enzyme conferred phosphomycin resistance to A. calcoaceticus . The polar effect of a rpoN mutation on the phosphomycin resistance level suggests that murZ is, in part, cotranscribed with rpoN . These observations confirm that A. calcoaceticus represents the first exceptin from a conserved genetic context of rpoN observed in several other Gram-negative bacteria.     

Analysis of the binding of p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime) to mitochondrial cytochrome c reductase.

Cytochrome c reductase is inhibited by p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime). CPMB-oxime induces a red-shift of the reduced spectrum of cytochrome b. The inhibitor blocks the oxidation of ubihydroquinone at the QP center of this enzyme in a non-competitive way. The binding stoichiometry equals one inhibitor molecule per Qp center. The apparent Kd in a red-shift assay was 6.9 +/- 0.6 microM. All binding characteristics analysed in this study were very similar to those of the E-beta-methoxyacrylate inhibitors, although the chemical structure is different from these inhibitors. This result is interpreted as a support for the inhibitory mechanism based on the model of a 'catalytic switch' proposed recently for the E-beta-methoxyacrylate inhibitors (MOA-inhibitors (Brandt and von Jagow, Eur. J. Biochem.     

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF^−/−, or p53^−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.     

Developmental Exposure to Fluoxetine Modulates the Serotonin System in Hypothalamus

The selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLU, Prozac®) is commonly prescribed for depression in pregnant women. This results in SSRI exposure of the developing fetus. However, there are knowledge gaps regarding the impact of SSRI exposure during development. Given the role of serotonin in brain development and its cross-talk with sex hormone function, we investigated effects of developmental exposure to pharmacologically relevant concentrations of FLU (3 and 30 nM (measured)) on brain neurotransmitter levels, gonadal differentiation, aromatase activity in brain and gonads, and the thyroid system, using the Xenopus tropicalis model. Tadpoles were chronically exposed (8 weeks) until metamorphosis. At metamorphosis brains were cryosectioned and levels of serotonin, dopamine, norepinephrine, and their metabolites 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid, and homovanillic acid were measured in discrete regions (telencephalon, hypothalamus and the reticular formation) of the cryosections using high-performance liquid chromatography. Exposure to 30 nM FLU increased the concentration of 5-hydroxyindoleacetic acid in hypothalamus compared with controls. FLU exposure did not affect survival, time to metamorphosis, thyroid histology, gonadal sex differentiation, or aromatase activity implying that the effect on the serotonergic neurotransmitter system in the hypothalamus region was specific. The FLU concentration that impacted the serotonin system is lower than the concentration measured in umbilical cord serum, suggesting that the serotonin system of the developing brain is highly sensitive to in utero exposure to FLU. To our knowledge this is the first study showing effects of developmental FLU exposure on brain neurochemistry. Given that SSRIs are present in the aquatic environment the current results warrant further investigation into the neurobehavioral effects of SSRIs in aquatic wildlife.     

“Self” and “Non-Self” in the Control of Phytoalexin Biosynthesis: Plant Phospholipases A2 with Alkaloid-Specific Molecular Fingerprints

The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the “self” plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate “self-made” from “foreign” alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins.