Structure-activity relationship of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone)

This paper describes the discovery of a novel free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone, 1), as a potent antioxidant agent against lipid peroxidation. The structure-activity relationship of edaravone indicated that lipophilic substituents were essential to show its lipid peroxidation-inhibitory activity. In vivo studies revealed that edaravone showed brain-protective activity in a transient ischemia model.     

Sojourn times and substitution rate at overdominant and linked neutral loci

The sojourn times until fixation of an overdominant allele were investigated based on the diffusion equation. Furthermore, the rate of accumulation of mutations, or the substitution rate, was predicted from the mean extinction time of a common overdominant allele. The substitution rate calculated theoretically agreed well with that determined by computer simulation. Overdominant selection enhances the polymorphism at linked loci, while its effect on the sojourn times and the substitution rate at linked loci has not been studied yet. To solve these problems, a model that assumed two linked loci, each with infinite alleles, was examined by computer simulation. A decrease in the recombination rate between two loci markedly changed the distribution of sojourn times of a neutral allele. Although overdominant selection obviously increased the sojourn times and the polymorphism at a linked locus, the rate of nucleotide substitution at the neutral locus was not influenced significantly even if complete linkage was assumed. These results suggest that, in regions containing overdominant genes, linked neutral loci will exhibit elevated levels of polymorphism, but their rate of molecular evolution remains that predicted by neutral theory.     

Computer simulation analysis suggests weak balancing selection operative at the MICA locus

A high degree of polymorphism has been reported at the major histocompatibility class I chain-related gene A (MICA) locus, which is located 46 kb away from HLA-Bin the human major histocompatibility complex (MHC) class I region. Although it is known that the polymorphisms at the conventional MHC class I loci have been maintained by balancing selection, it is unclear whether positive natural selection is also operative in maintaining the polymorphism at the MICA locus. In order to explain the degree of polymorphism at the MICA locus, a computer simulation study was carried out. The high degree of polymorphism at the MICA locus (heterozygosity and number of polymorphic residues) could not be explained solely by balancing selection at the HLA-B locus even if no recombination was assumed between MICA and HLA-B. Although there is no definite evidence indicating that balancing selection is operative at the MICA locus, our results suggest that the MICA gene is subject to weak balancing selection.     

Squid nerve sphingomyelin containing an unusual sphingoid base

A new methodology has been developed to determine sphingolipid structures by positive-ion fast atom bombardment tandem mass spectrometry (FAB-MS/MS). The method was verified by application to a structurally known glycosphingolipid, and then used in the structural study of an unusual sphingomyelin isolated from squid (Loligo pealei) nerve. Our previous study of this squid sphingomyelin indicated that the major base had a branched C(19) alkyl chain with three double bonds, two of which were conjugated. The positions of the branching as well as the double bonds of this base were unambiguously determined by directly comparing the product ion spectra of the long-chain base ion (LCB(+)) of two ceramides, one derived from squid nerve sphingomyelin and another, glucosylceramide, obtained from starfish spermatozoa. The latter served as the standard because the structure had already been determined by nuclear magnetic resonance (NMR). The precursor ion here was LCB(+), that is, [CH(2) - C(NH(2)) = CHR](+), rather than [M + H](+), where R represents the backbone hydrocarbon chain counting from C-4. The results clearly showed that the squid nerve base is identical to the base derived from starfish (Asterias amurensis), that is, 2-amino-9-methyl-4,8,10-octadecatriene-1,3-diol. This is the first report in which the detailed structure of a branched polyunsaturated sphingoid base was studied by tandem mass spectrometry without derivatization or the aid of NMR. The occurrence of such an unusual sphingoid base in various phyla and tissues suggests the conjugated polyunsaturated branched sphingoid base plays a significant role in animals.     

MHC (Major Histocompatibility Complex)-DRB Genes and Polymorphisms in Common Marmoset

A New World monkey, the common marmoset (Callithrix jacchus), will be used as a preclinical animal model to study the feasibility of cell and gene therapy targeting immunological and hematological disorders. For elucidating the immunogenetic background of common marmoset to further studies, in the present study, polymorphisms of MHC-DRB genes in this species were examined. Twenty-one Caja-DRB exon 2 alleles, including seven new ones, were detected by means of subcloning and the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) methods followed by nucleotide sequencing. Based on the alignment of these allele sequences, we designed two pairs of specific primers and established a PCR-SSCP method for DNA-based histocompatibility typing of the common marmoset. According to the family segregation data and phylogenetic analyses, we presumed that Caja-DRB alleles could be classified into five different loci. Southern blotting analysis also supported the existence of multiple DRB loci. The patterns of nucleotide substitutions suggests that positive selection operates in the antigen-recognition sites of Caja-DRB genes. Received: 18 February 2000 / Accepted: 17 May 2000     

Phylogenetic relationships of Salix (Salicaceae) based on rbcL sequence data

Nucleotide sequences of the chloroplast-encoded rbcL gene were used to examine phylogenetic relationships of the genus Salix together with other allied genera of the family Salicaceae. Phylogenetic analyses of rbcL sequences strongly suggest the monophyly of three commonly recognized genera (Chosenia, Salix, and Toisusu). Two monophyletic groups are recognized within the larger monophyletic group. They do not correspond with any infrageneric taxa proposed so far. With regard to character evolution, it is thought that the reduction of stamen number from more than two stamens to two might occur in at least three lineages and that fused bud scales evolved several times and/or the reverse evolution occurred from fused to free. Some types of pollen surfaces are considered to have evolved independently.     

Molecular cytogenetic analysis of eight inversion duplications of human chromosome 13q that each contain a neocentromere

Neocentromeres are fully functional centromeres that have arisen in previously noncentromeric chromosomal locations on rearranged chromosomes. The formation of neocentromeres results in the mitotic stability of chromosomal fragments that do not contain endogenous centromeres and that would normally be lost. Here we describe a unique collection of eight independent patient-derived cell lines, each of which contains a neocentromere on a supernumerary inversion duplication of a portion of human chromosome 13q. Findings in these patients reveal insight into the clinical manifestations associated with polysomy for portions of chromosome 13q. The results of FISH and immunofluorescent analysis of the neocentromeres in these chromosomes confirm the lack of alpha-satellite DNA and the presence of CENtromere proteins (CENP)-C, -E, and hMAD2. The positions of the inversion breakpoints in these chromosomes have been placed onto the physical map of chromosome 13, by means of FISH mapping with cosmid probes. These cell lines define, within chromosome 13q, at least three distinct locations where neocentromeres have formed, with five independent neocentromeres in band 13q32, two in band 13q21, and one in band 13q31. The results of examination of the set of 40 neocentromere-containing chromosomes that have thus far been described, including the 8 neocentromere-containing chromosomes from chromosome 13q that are described in the present study, suggest that chromosome 13q has an increased propensity for neocentromere formation, relative to some other human chromosomes. These neocentromeres will provide the means for testing hypotheses about sequence requirements for human centromere formation.     

An HR-induced tobacco peroxidase gene is responsive to spermine, but not to salicylate, methyl jasmonate, and ethephon

In Tobacco mosaic virus (TMV)-infected tobacco plants carrying the N resistance gene, a hypersensitive reaction or response (HR) occurs to enclose the virus in the infected tissue. Although a contribution of peroxidases to the resistance has been proposed, no evidence has been presented that tobacco peroxidase genes respond to HR. Here, we describe the HR-induced expression of a tobacco peroxidase gene (tpoxC1) whose induction kinetics were slightly different from those of acidic and basic tobacco pathogenesis-related (PR) protein genes. Interestingly, tpoxC1 was insensitive to the inducers of PR genes such as salicylic acid, methyl jasmonate, and ethephon. Spermine activated tpoxC1 gene expression at a low level and both acidic and basic PR gene expression at a considerably higher level. These results indicate that the induced expression of tpoxC1 is regulated differently from that of classical tobacco PR genes in the N gene-mediated self-defense system in tobacco plants.     

Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates

Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase.