Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis.     

Detection of 6-nitrotryptophan in proteins by Western blot analysis and its application for peroxynitrite-treated PC12 cells.

We have previously reported on the formation of 6-nitrotryptophan by the reaction of reactive nitrogen species with a tryptophan residue in human Cu, Zn-superoxide dismutase (SOD) (F. Yamakura et al., J. Biochem. 138 (2005) 57-69). Here, we report on the preparation of anti-6-nitrotryptophan antiserum by using synthesized 6-nitrotryptophan-conjugated keyhole limpet hemocyanin as an antigen and the purification of the antibody by using a 6-nitrotryptophan-conjugated affinity column. The purified antibody was immunoreactive with 6-nitrotryptophan residue containing Cu, Zn-SOD but not immunoreactive with Cu, Zn-SOD, Mn-SOD, bovine serum albumin, and 3-nitrotyrosine residue containing Mn-SOD. Nitro group of 6-nitrotryptophan was reduced by sodium hydrosulfite to form 6-aminotryptophan as a major product. The reduced 6-nitrotryptophan residues lost its immunoreactivity with the antibody. We detected different immunoreactive bands between using antibody for 6-nitrotryptophan residues and that for 3-nitrotyrosine residues in crude extracts of neuron-like PC12 cells treated with peroxynitrite by a Western blot analysis. Western blot analysis for two-dimensional gel electrophoresis showed nine intensively stained immunoreactive spots for 6-nitrotryptophan residues in the peroxynitrite-treated PC12 cells, which were subjected to trypsin digestion and LC-ESI-MS/MS analysis. We identified M2 pyruvate kinase, elongation factor 2, mitochondrial aconitase, pyruvate carboxylase, and heat shock protein HSP90alpha as candidates for 6-nitrotryptophan residues containing proteins, with peptide coverage over 10%, in crude extracts of peroxynitrite-treated PC12 cells.     

The effects of ionic strength on the protein conformation and the fluidity of porcine intestinal brush border membranes. Fluorometric studies using N-[7-dimethylamino-4-methylcoumarinyl]maleimide and pyrene

The effects of ionic strength on the conformation around the SH groups of the proteins and the lipid fluidity of porcine intestinal brush border membranes were studied using two fluorescent dyes, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM) and pyrene. The extent of DACM labeling to the SH groups of the membrane proteins was accelerated depending on the KCl concentrations in medium. A quenching study of DACM-labeled membranes with acrylamide showed that the proximity of the quencher to the fluorescence-labeled SH groups in the membrane proteins is increased with increasing ionic strength of medium. An implication of the conformational changes around SH groups in the membrane proteins with increase of ionic strength was also obtained from the stimulation of guanidine effect on the fluorescence parameters of DACM-labeled membranes by addition of KCl. On the other hand, the results of the quenching study with KI, excimer fluorescence, and polarization measurements of pyrene-labeled membranes suggested an increase of membrane fluidity on addition of KCl to medium. The temperature dependence of polarization of the complex strongly suggested that the rotational freedom of pyrene molecules embedded into the lipid layers of the membranes is increased by addition of KCl. In fact, the harmonic means of the rotational relaxation times of pyrene molecules in the membranes with and without 100 mM KCl were estimated to be about 2900 and 9000 ns at 25 degrees C, respectively. Based on these results, the salt-induced alterations of the conformation in the vicinity of the bound dyes of the membrane proteins and of the membrane fluidity are discussed.     

Structure and Characterization of a Gene for Light-Harvesting Chi a/b Binding Protein from Rice

The gene for the light-harvesting chlorophyll a/b binding proteinfrom rice was cloned and se-quenced. The clone contains a 798-bpcoding sequence, which is identical to that of a cDNA for typeI LHCPII (Matsuoka 1990), and its 5'- and 3'-flanking regions.The coding region of this gene is not interrupted by interveningsequences, as reported for type I genes from other plants. Inthe 5'-flanking region, typical TATA and CAAT boxes are located30 and 92 bp upstream from the capping site (positions –30and –92), respectively. A putative phytochrome-responsiveelement (AAGATAAGG) is located at position –65 betweenthe TATA and CAAT boxes. Comparison of sequences in the 5'-flankingregions between this gene and genes for LHCPII from other gramineousplants indicates that the rice sequence has no apparent homologyto that of wheat. However, the rice sequence is highly homologousto the maize sequence, not only around the TATA and CAAT boxesbut also in regions further upstream. To investigate the promoter activity of the 5'-flanking regionof the gene, a chimeric gene was constructed by fusing the 5'-flankingregion to the coding sequence for ß-glucuronidase(GUS), and this chimeric gene was introduced into tobacco. Thehighest activity of GUS was observed in leaf tissue, indicatingthat the 5'-flanking region of the gene can act as a promoterin an organ-specific manner in tobacco. Histochemical analysisin situ was also performed to determine where GUS activity wasexpressed. The highest activity was found in leaf mesophyllcells. High activity was also observed in the vascular systemof stems and petioles, and low activity was found in root tissue. (Received August 20, 1990; Accepted January 21, 1991)     

Behavior of cell aggregate of Carthamus tinctorius L. cultured cells and correlation with red pigment formation

The formation processes of Carthamus tinctorius cell aggregates in a growth medium and the correlation of red pigment formation with cell aggregate sizes were investigated. About 80% of cell aggregates in the growth medium were > 1.00 mm in size. The growth rate of large cell aggregates was more rapid than that of small cell aggregates. Most cell aggregates > 0.50 mm in size became larger or smaller than their original sizes during the culture. A high level of red pigment formation was observed when cell aggregates obtained by the preculture using cell aggregates < 1.00 mm in size were cultured in the production medium.     

Vasorelaxant activity of 2-substituted 6-nitro-2H-1-benzopyran-4-carbothioamide K channel openers

Synthesis and vasorelaxant activity of 2-substituted 6-nitro-2H-1-benzopyran-4-carbothioamides 5 are described. Potent smooth muscle relaxant activity was displayed by 5c, 5h, and 5i.     

Vasorelaxant activity of 2-fluoromethylbenzopyran K channel openers

Synthesis and vasorelaxant activity of 2-fluoromethylbenzopyrans are explained. These 2-fluoromethyl derivatives showed a potent smooth muscle relaxant activity.     

Synthesis and vasorelaxant activity of N-imino-2-(benzopyran-4-yl)pyridine K channel openers

The synthesis and vasorelaxant activity of N-imino-2-(benzopyran-4-yl)pyridines are described. Some of these compounds displayed potent smooth muscle relaxant activity.