Determination of plasma [18F]-6-fluorodopa during positron emission tomography: elimination and metabolism in carbidopa treated subjects

An investigation of the metabolism of [18F]-6-fluorodopa (FDOPA) given to carbidopa treated subjects for scanning by positron emission tomography (PET) has been carried out by analysis of plasma. Reverse phase ion pair HPLC and alumina extraction were employed to fractionate and identify the [18F]-labelled compounds of plasma over a two hour period. During this time, the plasma levels of both total 18F and FDOPA decreased as a bi-exponential function of time. The rates of 18F, but not FDOPA, elimination were observed to decrease with age. In addition to FDOPA, only one other major peak of radioactivity was resolved by HPLC. Identification of this compound as the O-methylated derivative of FDOPA (MeFDOPA) is based on its shared HPLC elution time with in vitro synthesized O-[methyl-14C]-FDOPA. The ratio of the concentration of MeFDOPA to FDOPA (MeFDOPA/FDOPA) in plasma increased linearly with time, and the slope of this linear relationship decreased with the age of the individual.     

Stimulation of V(D)J recombination by histone acetylation

V(D)J recombination assembles functional immunoglobulin and T cell receptor genes from individual gene segments [1]. A common recombination mechanism, initiated by the proteins RAG1 and RAG2 at conserved recombination signal sequences (RSSs), operates at all rearranging loci [2] [3]. It has been proposed that the key regulator of the reaction is 'accessibility' of the RSS within chromatin [4]. Recently, the packaging of RSSs into nucleosomes was shown to inhibit initiation of V(D)J recombination [5] [6]. Nevertheless, the tight tissue specificity of regulation cannot be explained by nucleosome-mediated repression alone because a significant fraction of RSSs would be predicted to lie in linker regions between nucleosomes. Therefore, some aspect of the regulation of the recombination reaction must rely on the disruption of higher-order chromatin structure. Here, we report that histone acetylation directly stimulates the recombination reaction in vivo in the correct cell- and stage-specific manner. Neither expression of RAG genes nor activity of RAG proteins was increased by acetylation. Furthermore, histone acetylation failed to overcome nucleosome-mediated repression of RSS recognition and cleavage in vitro. Our data suggest a role for histone acetylation in stimulating recombination in vivo through disruption of higher-order chromatin structures.     

A statistical evaluation of the reproducibility of micronucleus, sister-chromatid exchange, thioguanine-resistance and ouabain-resistance assays in V79 cells exposed to ethyl methanesulfonate and 7,12-dimethylbenz[a]anthracene

In contrast to the "validation" of short-term in vitro genotoxicity assays by concordance with the rodent cancer bioassay, the present report describes the multiple replication of 4 short-term tests with V79 cells (micronucleus assay, MN; sister-chromatid exchange, SCE; ouabain resistance. OUR; and thioguanine resistance, TGR) within the same assay system following exposure to each of two genotoxins, ethyl methanesulfonate (direct acting) and 7,12-dimethylbenz[a]anthracene (indirect acting). Reproducibility, proportion of genotoxins correctly identified, and proportion of non-genotoxins correctly identified by each test were each determined statistically. Decision rules were formulated to declare a positive response in each assay, and overall accuracy of each was determined. Statistical analysis of the data, obtained under standardized test conditions, showed that for these two chemicals SCE identified 100% of genotoxins and 86% of non-genotoxins, with overall accuracy of prediction of 93%; TGR identified 98% of genotoxins and 74% of non-genotoxins, with overall accuracy of 86%; MN identified 78% of genotoxins and 84% of non-genotoxins, with overall accuracy of 81%; while OUR indicated 100% of genotoxins, but only 50% of non-genotoxins, and only 76% overall accuracy. The results suggested that the best overall accuracy of classification with the V79 assay system could be achieved by measurement of SCE in combination with thioguanine resistance.     

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.     

Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA.      

In acute pathogenic SIV infection plasmacytoid dendritic cells are depleted from blood and lymph nodes despite mobilization

Background Plasmacytoid dendritic cells (pDC) are depleted from blood of individuals with HIV infection associated with progression to disease. It has been postulated but not proven that pDC accumulate in lymph nodes and induce sustained immune activation characteristic of disease. Methods The dynamics of the pDC response to acute pathogenic SIV infection of rhesus macaques were studied using methods to track recently divided cells. Results pDC were lost from blood and lymph nodes in acute SIV infection despite rapid mobilization and recruitment. pDC had a low frequency of infection, were uniformly activated and had increased levels of apoptosis, while maintaining normal function. Conclusions pDC mobilization into blood and lymph nodes in acute SIV infection does not keep pace with excessive pDC loss through activation and apoptosis. The depletion of pDC from lymphoid tissues in acutely infected rhesus macaques does not support a pathogenic role for pDC in disease.     

PDA: Pooled DNA analyzer

Background  

Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer), to analyze pooled DNA data.     

A Gene Cluster Encoding Steps in Conversion of Naphthalene to Gentisate in Pseudomonas sp. Strain U2

Pseudomonas sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. The genes for naphthalene dioxygenase were cloned from the plasmid DNA of strain U2 on an 8.3-kb BamHI fragment. The genes for the naphthalene dioxygenase genes nagAa (for ferredoxin reductase), nagAb (for ferredoxin), and nagAc and nagAd (for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations and by nucleotide sequencing. The genes for nagB (for naphthalene cis-dihydrodiol dehydrogenase) and nagF (for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between nagAa and nagAb were two open reading frames, homologs of which have also been identified in similar locations in two nitrotoluene-using strains (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 181:57–61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926–4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet. Eng. 19:257–269, 1997). Recombinant Escherichia coli strains with plasmids carrying this region were able to convert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance. The first open reading frame, designated nagG, encodes a protein with characteristics of a Rieske-type iron-sulfur center homologous to the large subunits of dihydroxylating dioxygenases, and the second open reading frame, designated nagH, encodes a protein with limited homology to the small subunits of the same dioxygenases. Cloned together in E. coli, nagG, nagH, and nagAb, were able to convert salicylate (2-hydroxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore encode a salicylate 5-hydroxylase activity. Single-gene knockouts of nagG, nagH, and nagAb demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunits of salicylate 5-hydroxylase linked to an electron transport chain consisting of NagAb and NagAa, although E. coli appears to be able to partially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization. However growth on both naphthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2. Sequence comparisons suggest that the novel gene order nagAa-nagG-nagH-nagAb-nagAc-nagAd-nagB-nagF represents the archetype for naphthalene strains which use the gentisate pathway rather than the meta cleavage pathway of catechol.