The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps in brassinosteroid biosynthesis.

dwarf4 (dwf4) mutants of Arabidopsis display a dwarfed phenotype due to a lack of cell elongation. Dwarfism could be rescued by the application of brassinolide, suggesting that DWF4 plays a role in brassinosteroid (BR) biosynthesis. The DWF4 locus is defined by four mutant alleles. One of these is the result of a T-DNA insertion. Plant DNA flanking the insertion site was cloned and used as a probe to isolate the entire DWF4 gene. Sequence analysis revealed that DWF4 encodes a cytochrome P450 monooxygenase with 43% identity to the putative Arabidopsis steroid hydroxylating enzyme CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM. Sequence analysis of two other mutant alleles revealed deletions or a premature stop codon, confirming that DWF4 had been cloned. This sequence similarity suggests that DWF4 functions in specific hydroxylation steps during BR biosynthesis. In fact, feeding studies utilizing BR intermediates showed that only 22alpha-hydroxylated BRs rescued the dwf4 phenotype, confirming that DWF4 acts as a 22alpha-hydroxylase.     

Partial Characterization of a 17 kDa Acidic Protein, EFP, Induced by Thiocarbamate in the Early Flowering Phase in Asparagus Seedlings

Asparagus officinalis seedlings treated with either thiocarbamatesor carbamates are induced to flower within 2-3 weeks of application.SDS-PAGE and 2-D PAGE resolutions of total soluble proteinsof treated seedlings showed the accumulation of an acidic 17kDa polypeptide. Partialmino acid sequence of the acidic proteinshowed homology to the wound-responsive A. officinalisPR 1,AoPRl. We named this protein EFP, early flowering protein, owingto its advanced appearance during the early transition fromvegetative to floral phase. Carbamates and thiocarbamates thatinduced more than 80% flowering rates corresponded with increasedlevel of EFP while compounds that are weak flower inducers showedreduced amount of EFP. The observation thus denned the correlationshipof EFP to variable flowering rates. (Received May 27, 1996; Accepted July 11, 1996)     

A new approach for improving cordycepin productivity in surface liquid culture of Cordyceps militaris using high‐energy ion beam irradiation

Aims: To obtain a higher cordycepin production using Cordyceps militaris mutant obtained by a new mutagenesis technique called ‘ion beam’. Methods and Results: Successful irradiation of C. militaris NBRC 9787 by a proton beam with high energy was performed, and 30 classes of 8‐azaadenine‐ and 28 classes of 8‐azaaguanine‐resistant mutants were obtained on mutant screening, of which seven classes were selected as promising preliminary mutants having an antibacterial ability as an index of cordycepin production. In a surface liquid culture technique, some of the 8‐azaadenine‐resistant mutants gave a better performance for the cordycepin productivity; in contrast, among the 8‐azaaguanine‐resistant mutants, it was shown that mutant no. G81‐3 was much better than the control in the metabolic rate of glucose and the cordycepin productivity. In primary optimization using the enriched medium, the cordycepin production was 3·1 and 1·8 g l^?1 on 21‐day culture for mutant no. G81‐3 and the control, respectively. The cordycepin production obtained by the mutant was 72% more than the control. Conclusions: The mutant obtained by proton beam irradiation had higher productivity of cordycepin than that of the control. Significance and Impact of the Study: The mutant obtained by irradiation had a superior production performance of cordycepin, and therefore, it could be used in the realm of applied industrial biotechnology for the large‐scale production of cordycepin.     

Klotho-related protein is a novel cytosolic neutral beta-glycosylceramidase

Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-insensitive neutral glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and rat brains, and human fibroblasts. The candidates for the enzyme were assigned to the Klotho (KL), whose family members share a beta-glucosidase-like domain but whose natural substrates are unknown. Among this family, only the KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when expressed in CHOP cells, in which Myc-tagged KLrP was exclusively distributed in the cytosol. In addition, knockdown of the endogenous KLrP by small interfering RNA increased the cellular level of GlcCer. The purified recombinant KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl derivatives, but each k(cat)/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6A resolution revealed that KLrP is a (beta/alpha)8 TIM barrel, in which Glu(165) and Glu(373) at the carboxyl termini of beta-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. The substrate-binding cleft of the enzyme was occupied with palmitic acid and oleic acid when the recombinant protein was crystallized in a complex with glucose. GlcCer was found to fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer.     

Synthesis and pharmacological profile of serofendic acids A and B

We present efficient syntheses of serofendic acids A and B (SA-A and SA-B), novel neuroprotective substances isolated from fetal calf serum. Biological and pharmacological evaluation showed that SA-A and SA-B have potent protective action against glutamate-induced neurotoxicity, but do not interact directly with glutamate receptors. A pharmacokinetic study showed that they have good oral bioavailability in rats. The results indicate that SA-A and SA-B are potential lead compounds for candidate drugs to treat various neurological disorders.     

Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family

Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.     

Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.     

A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud

Mammal-fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements.