New, highly amino-substituted dextran or aminodextran (hereafter denoted Amdex) of various sizes between about 20 and 1000 kDa molecular mass and degrees of amino-substitution between 7 and 40% were prepared and characterized by elemental analyses and polyacrylamide gel electrophoresis. These aminodextrans together with others commercially available were shown by static light scattering, viscosity, and refractive index measurements to adopt a globular structure in aqueous salt solutions. Antibody and fluorescent protein dye, phycoerythrin, or its tandems with cyanin 5. 1 and TEXAS RED, were covalently conjugated to the aminodextrans. The conjugates contained multiple dye molecules and were shown by dynamic light scattering and scanning electron microscopy to assume either globular structure or aggregates thereof. Streptavidin could be substituted for antibody to prepare streptavidin-aminodextran-PE conjugates, which were then used with biotinylated antibody to label subpopulations of white blood cells. The conjugates yielded up to 20-fold amplification of fluorescence intensity over direct antibody-dye conjugates in labeling white blood cells for flow cytometry.

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987 Group I introns include nuclear rRNA introns of Tetrahymena spp. and Physarum polycephalum, most fungal mitochondrial DNA introns, some chloroplast introns, and introns in T-even bacteriophages (5). All group I introns appear to use the same splicing mechanism first elucidated for the Tetrahymena thermophila nuclear rRNA intron by T. R. Cech and co-workers. This mechanism involves two sequential transesterification reactions: addition of guanosine to the 5' end of the intron coupled to cleavage at the 5' splice site and exon ligation coupled to cleavage at the 3' splice site (5). Some group I introns, including the Tetrahymena nuclear rRNA intron, some mitochondrial DNA introns in Neurospora crassa and Saccharomyces cerevisiae, and introns in T-even bacteriophage, are efficiently self splicing in vitro (5). Self-splicing indicates that both the structural information and catalytic activities required for splicing are contained in the structure of the intron RNAs. The basic outline of the catalytically active structure of group I introns has been elucidated by phylogenetic comparisons, analysis of in vivo and in vitro mutants, and direct RNA structure analysis (5). All a core secondary structure that includes base pairing between short sequence elements P3[5'], P, Q, R, P3[3'], and S. Group I introns also contain an internal guide sequence that base pairs with flanking sequences in the 5' exon to position the 5' splice site for splicing and may also play a role in positioning the 3' splice site (4, 5). The internal region of the intron, including the core structure, possesses the catalytic activities that cleave at the 5' splice site and add guanosine to the 5' end of the intron (35). In those group I introns that are self-splicing in vitro, the catalytically active structure must be favored relative to alternative structures of deproteinized precursor RNAs. Although some group I introns are self-splicing in vitro, genetic analysis has shown that most, if not all, group I introns are dependent upon proteins for splicing in vivo. The most likely hypothesis is that these proteins function to fold the RNA into the catalytically active conformation. We showed previously that N. crassa cob intron 1, which is efficiently self-splicing in vitro, is dependent on the protein encoded by the cyt-18 gene for splicing in vivo (6,12). The N. crassa mitochondrial large rRNA intron has been shown to require proteins for splicing both in vivo and in vitro (13).The proteins required for splicing group I introns can be divided into three classes: (i) maturases, a family of structurally related proteins encoded within some, but not all, group I introns (26, 36); (ii) intron-specific proteins encoded by nuclear genes, e.g., the CBP2 and MRS1 proteins of S. cerevisiae, whic...

No abstract

No abstract

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.

Background: The need to implement robust biomarkers in clinical trials has never been greater, and such efforts can be easily compromised by reagent instability or simple human error during assay setup. Many biotechnology and pharmaceutical companies are introducing efforts to conduct biomarker studies under more rigorous settings, and the use of plates or tubes pre-loaded with stimulation or staining reagents could be of value for studies that involve flow cytometry.Methods: Five reagents lyophilized from ethanol or CHAPS buffer stock solution of phorbol 12-myristate 13-acetate (PMA) and ionomycin were benchmarked against standard DMSO liquid formulation for their stimulation equivalency. The median fluorescence intensity of phosphorylated ribosomal protein S6 in lymphocytes was assessed on a BD FACSCalibur TM . Results: We demonstrate here that tubes pre-loaded with lyophilized versions of the liquid reagents can provide equivalent stimulation in healthy volunteer specimens.Conclusions: The value of this approach is that it safeguards against omission or erroneous addition of bulk liquid formulations of PMA and ionomycin to the reaction vessel (i.e., plate or tube) and also lends itself to extended stability/shelf-life of these reagents. On the basis of this initial success, we plan to expand our evaluation of lyophilized reagents so that they can be incorporated into our clinical biomarker campaigns as appropriate. V C 2009 Clinical Cytometry Society

No abstract

No abstract