Production and identification of bityrosine in horseradish peroxidase-H2O2-tyrosine system

Production of bityrosine (2,2'-dihydroxy-5,5'-bis (beta-carboxy-beta-aminoetyl)-diphenyl) was established in horseradish peroxidase-H2O2-tyrosine system at pH 9.2 by mass and NMR spectrometries.     

Closely Linked Polymorphic Markers for Determining the Autosomal Dominant Allele (Cy) in Rat Polycystic Kidney Disease

The Han:SPRD strain is an SD-background strainknown to be a model of polycystic kidney disease (PKD)expressed through an autosomal dominant gene (Cy).However, different genotypes of this strain cannot be identified in the neonatal period. First, toestablish an accurate method of determining thegenotypes (Cy/Cy, Cy/+, +/+) which cause differentdisease progressions, we used polymorphic markers on rat chromosome 5. PCR products of tissue DNAtemplated with D5Rat9 showed distinct patterns onelectrophoresis indicating three genotypes. Second, todetermine whether the same locus plays a major role inexpressing PKD, we performed linkage analyses in a [BN X(BN X Han:SPRD)F₁] backcross. Cy/Cy and Cy/+also caused PKD in a BN background. In this backcross,we discovered that D5Rat11 is located closer to the Cy locus than D5Mgh10, which is regardedas one of the closest loci. We conclude that D5Rat9 andD5Rat11 are useful markers for determining the presenceof the Cy allele, which is regarded as the gene responsible for PKD.     

A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions

Glc has hormone-like functions and controls many vital processes through mostly unknown mechanisms in plants. We report here on the molecular cloning of GLUCOSE INSENSITIVE1 (GIN1) and ABSCISIC ACID DEFICIENT2 (ABA2) which encodes a unique Arabidopsis short-chain dehydrogenase/reductase (SDR1) that functions as a molecular link between nutrient signaling and plant hormone biosynthesis. SDR1 is related to SDR superfamily members involved in retinoid and steroid hormone biosynthesis in mammals and sex determination in maize. Glc antagonizes ethylene signaling by activating ABA2/GIN1 and other abscisic acid (ABA) biosynthesis and signaling genes, which requires Glc and ABA synergistically. Analyses of aba2/gin1 null mutants define dual functions of endogenous ABA in inhibiting the postgermination developmental switch modulated by distinct Glc and osmotic signals and in promoting organ and body size and fertility in the absence of severe stress. SDR1 is sufficient for the multistep conversion of plastid- and carotenoid-derived xanthoxin to abscisic aldehyde in the cytosol. The surprisingly restricted spatial and temporal expression of SDR1 suggests the dynamic mobilization of ABA precursors and/or ABA.     

A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections

The immunodeficient mice transplanted with human hepatocytes are available for the study of the human hepatitis viruses. Recently, human hepatocytes were also successfully transplanted in herpes simplex virus type-1 thymidine kinase (TK)-NOG mice. In this study, we attempted to infect hepatitis virus in humanized TK-NOG mice and urokinase-type plasminogen activator-severe combined immunodeficiency (uPA–SCID) mice. TK-NOG mice were injected intraperitoneally with 6 mg/kg of ganciclovir (GCV), and transplanted with human hepatocytes. Humanized TK-NOG mice and uPA/SCID mice were injected with hepatitis B virus (HBV)- or hepatitis C virus (HCV)-positive human serum samples. Human hepatocyte repopulation index (RI) estimated from human serum albumin levels in TK-NOG mice correlated well with pre-transplantation serum ALT levels induced by ganciclovir treatment. All humanized TK-NOG and uPA–SCID mice injected with HBV infected serum developed viremia irrespective of lower replacement index. In contrast, establishment of HCV viremia was significantly more frequent in TK-NOG mice with low human hepatocyte RI (<70%) than uPA–SCID mice with similar RI. Frequency of mice spontaneously in early stage of viral infection experiment (8 weeks after injection) was similar in both TK-NOG mice and uPA–SCID mice. Effects of drug treatment with entecavir or interferon were similar in both mouse models. TK-NOG mice thus useful for study of hepatitis virus virology and evaluation of anti-viral drugs.     

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (S ^a, S ^b, S ^c, S ^d). We previously reported the cDNA cloning of three of these alleles, namely S ^b, S ^c and S ^d. In this paper we report the cloning and DNA sequence analysis of the S ^a allele. The S^a-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (S^b, S^c, and S^d) and this similarity was lower than that observed among the S^b, S^c and S^d-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; S ^a (602 bp), S ^b (1083 bp), S ^c (221 bp) and S ^d (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the S ^a- and S ^b-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the S ^a-allele and 556 bp in the S ^b-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage. Received: 21 June 1999 / Accepted: 15 November 1999