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.     

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.     

Subunit II of cytochrome c oxidase from Paracoccus denitrificans. DNA sequence, gene expression and the protein

Cytochrome c oxidase from the bacterium Paracoccus denitrificans, while being related to the mitochondrial enzyme in many ways, consists of only two to three different subunits. For the identification of its genes, a Paracoccus DNA library was constructed and screened with specific antibodies for expression of cloned inserts in E. coli. A positive clone expressing immunoreactive products in the molecular mass region of authentic subunit II revealed a high homology of its DNA-deduced amino acid sequence with subunit II sequences of the mitochondrial oxidases; several typical features, such as the transmembrane folding pattern and the presumed copper-binding site, are highly conserved between prokaryotic and mitochondrial polypeptides. A comparison with peptide sequencing data of the purified subunit established the presence of a characteristic N-terminal extension as well as a longer C terminus in the initial translation product of the Paracoccus subunit; by mass spectroscopy, the first N-terminally blocked residue of the mature polypeptide was identified as a pyroglutamate. No code abnormalities, but a highly specific codon usage were observed; no evidence for a localization of the subunit I gene directly adjacent to this gene has been obtained.     

Effect of paclobutrazol on accumulation of organic acids and total phenols in apple wood

Apple trees (Malus domestica Borkh Spartan grafted on MM 106 rootstock) planted in 1976 in an orchard at Beltsville, Maryland, were treated with paclobutrazol (2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-1,2,4-triazol-1-yl-pentan-3-ol) using a foliage spray in 1982 and by trunk banding in 1983. Paclobutrazol did not inhibit shoot growth in 1983; however, shoot growth was significantly retarded in 1984. Increases in organic acids, including succinic, malic, citric, and quinic, and also in total phenols, occurred in wood produced in 1983 on paclobutrazol-treated trees when growth was not inhibited and in wood produced in 1984 when growth was inhibited. The organic acid content of both paclobutrazol-treated and untreated wood tended to decrease from the winter dormant period through growth resumption in the spring. However, the content of total phenols remained nearly the same throughout this sampling period.Mention of a trademark, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae

The ATP synthase complex of Klebsiella pneumoniae (KF₁F₀) has been purified and characterized. SDS-gel electrophoresis of the purified F₁F₀ complexes revealed an identical subunit pattern for E. coli (EF₁F₀) and K. pneumoniae. Antibodies raised against EF₁ complex and purified EF₀ subunits recognized the corresponding polypeptides of EF₁F₀ and KF₁F₀ in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA 9-amino-6-chloro-2-methoxyacridine - DCCD N,N-dicyclohexylcarbodiimide - FITC fluorescein isothiocyanate - SDS sodium dodecyl sulfate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole     

Tissue susceptibility factors in cadmium carcinogenesis

Recently, in two separate studies we have observed cadmium (Cd)-induction of prostatic tumors (PT) in rats. Cd (sc or im) at doses nontoxic to the testes markedly increased PT formation (2.5 μmol/kg, sc, 8 PT/29 exposed, 28%; 30 μmol/kg, im, 11/26, 42%; control 14/127, 11%). The administration of zinc (Zn; 1 mmol/kg, sc, at ?6, 0 and +18 h) to prevent testicular toxicity and tumors from Cd (30 μmol/kg, sc, 0 h) also resulted in an elevated incidence of PT (8/27, 30%). The nature of the metal-binding proteins in the prostate has not been defined, although metallothionein (MT), a low M_r Cd-binding protein that confers tolerance to Cd, is deficient in other target tissues of Cd carcinogenesis, such as the rat testes. Using a technique that extracts MT from liver, a low-M_r Cd-binding protein was extracted from both ventral (VP) and dorsal prostate (DP) and isolated by gel filtration. In contrast to the two forms of rat MT, reverse phase HPLC of VP and DP extract eluted 1 and 5 forms, respectively. The amino acid compositions of the VP and DP proteins were quite distinct from MT, with much less cys than MT and the presence of residues not found in MT (leu, tyr, phe). Thus Cd-induction of PT appears to be dependent on functional testes and, as is the case with Cd-induced testicular formation, appears to be associated with a deficiency of MT.