Molecular and Biochemical Characterization of Three Anthocyanin Synthetic Enzymes from Gentiana triflora

Full length cDNA clones of flavonoid 3',5'-hydroxylase, dihydroflavonol4-reductase and flavonoid 3-glucosyltransferase were clonedfrom petals of Gentiana triflora. Their sequences were homologousto counterparts from other plants. Flavonoid 3',5'-hydroxylaseand flavonoid 3-glucosyltransferase were enzymatically characterizedby expressing cDNAs in heterologous expression systems. (Received May 21, 1996; Accepted June 4, 1996)     

Intervention of somatic mutational events in vivo by a germline defect at the adenine phosphoribosyltransferase locus

Both germline and somatic mutations are known to affect phenotypes of human cells in vivo. In previous studies, we cloned mutant peripheral blood T cells from germline heterozygous humans for adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) deficiency and found that approximately 1.3 × 10^–4 peripheral T cells had undergone in vivo somatic mutations. Loss of heterozygosity (LOH) was the major cause of the mutations at the APRT locus since approximately 80% of the mutant T cell clones exhibited loss of normal alleles. In the present study, we identified three heterozygous individuals for APRT deficiency (representing two separate families), in whom none of the somatic mutant cells exhibited LOH at the APRT locus. The germline mutant APRT alleles of these heterozygotes from two unrelated families had the same gross DNA abnormalities detectable by Southern blotting. None of the germline mutant APRT alleles so far reported had such gross DNA abnormalities. The data suggest that the germline mutation unique to these heterozygous individuals is associated with the abrogation of LOH in somatic cells. The absence of LOH at a different locus has already been reported in vitro in an established cell line but the present study describes the first such event in vivo in human individuals. Received: 10 May 1996     

Cloning and characterization of elastase structural gene from Pseudomonas aeruginosa IFO 3455

An 8.3 Kb DNA fragment was cloned from Pseudomonas aeruginosa IFO 3455. This fragment-containing Escherichia clone, pEL2, produced a high level of elastase activity. A smaller EcoRI-KpnI fragment was subcloned into pUC118 and E. coli HB101 was transformed with the plasmid. A deletion mutant clone was also constructed in the same bacteria. These deletion mutants were tested for elastase activity and it became clear that the full length of the elastase gene was 1.0-1.3 Kb. DNA sequencing analysis revealed that this DNA fragment contains the DNA sequence coding N-terminal amino acid sequence of the elastase protein.     

Synthesis and evaluation of bioactive naphthoquinones from the Brazilian medicinal plant, Tabebuia avellanedae

A series of naphthoquinones based on the naphtho[2,3-b]furan-4,9-dione skeleton such as (−)-5-hydroxy-2-(1′-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (1) and its positional isomer, (−)-8-hydroxy-2-(1′-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (2), which are secondary metabolites found in the inner bark of Tabebuia avellanedae, were stereoselectively synthesized and their biological activities were evaluated in conjunction with those of their corresponding enantiomers. Compound 1 exhibited potent antiproliferative effect against several human tumor cell lines, but its effect against some human normal cell lines was much lower than that of mitomycin. On the other hand, its enantiomer (R)-1 was less active toward the above tumor cell lines than 1. The antiproliferative effect of 2 against all tumor cell lines was significantly reduced. These results indicated the presence of the phenolic hydroxy group at C-5 is of great important for increasing antiproliferative effect. In addition, 1 also showed higher cancer chemopreventive activity than 2, while there were no significant differences between 1 and 2 in antimicrobial activity. Both compounds displayed modest antifungal and antibacterial activity (Gram-positive bacteria), whereas they were inactive against Gram-negative bacteria.     

Lipid Peroxidation by the [Peroxidase/H2O2/Phenolic] System

Linoleic acid was oxygenated by horseradish peroxidase (EC 1.11.1.7 [EC] )in the presence of phenolics. The phenolics effective for thissystem had substituents at the P-position. The peroxidase-dependentlipid peroxidation produced reaction products similar to thoseproduced by lipoxygenase (EC 1.13.1.13 [EC] ) under the same conditions.Positional isomers of the reaction products were identifiedas 13-hydroperoxy-9, lloctadecadienoic acid and 9-hydroperoxy-10,12-octadecadienoicacid. (Received November 15, 1986; Accepted March 19, 1987)     

Sphingosine 1-Phosphate Receptor 4 Uses HER2 (ERBB2) to Regulate Extracellular Signal Regulated Kinase-1/2 in MDA-MB-453 Breast Cancer Cells

We demonstrate here that the bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P₄) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. This was based on several lines of evidence. First, the S1P stimulation of ERK-1/2 was abolished by JTE013, which we show here is an S1P_2/4 antagonist and reduced by siRNA knockdown of S1P₄. Second, the S1P-stimulated activation of ERK-1/2 was almost completely abolished by a HER2 inhibitor (ErbB2 inhibitor II) and reduced by siRNA knockdown of HER2 expression. Third, phyto-S1P, which is an S1P₄ agonist, stimulated ERK-1/2 activation in an S1P₄- and HER2-dependent manner. Fourth, FTY720 phosphate, which is an agonist at S1P_1,3,4,5 but not S1P₂ stimulated activation of ERK-1/2. Fifth, S1P stimulated the tyrosine phosphorylation of HER2, which was reduced by JTE013. HER2 which is an orphan receptor tyrosine kinase is the preferred dimerization partner of the EGF receptor. However, EGF-stimulated activation of ERK-1/2 was not affected by siRNA knockdown of HER2 or by ErbB2 (epidermal growth factor receptor 2 (or HER2)) inhibitor II in MDA-MB-453 cells. Moreover, S1P-stimulated activation of ERK-1/2 does not require an EGF receptor. Thus, S1P and EGF function in a mutually exclusive manner. In conclusion, the magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P₄ may have an important role in breast cancer progression.     

Induction and Secretion of Pathogenesis-Related Proteins by Salicylate or Plant Hormones in Tobacco Suspension Cultures

Pathogenesis-related proteins (PR proteins), that are inducedin tobacco leaves in hypersensitive response to infection withtobacco mosaic virus (TMV) or by treatment with chemicals, werefound to be also inducible in a dedifferentiated system, tobaccosuspension culture. Quantitative determination of these proteinsusing anti PR 1a IgG showed that their increase started at aboutthe end of cell growth period and that their production couldbe enhanced by the addition of potassium salicylate, Eosin Yellowishand plant hormones (GA3, IAA and 2,4-D). The production dependedon the concentration of the chemical inducer and the cell lineused. In BY-2 cell line, PR proteins amounted to 12 µgat day 5 and then increased exponentially with time, reaching280 µg or 70 µg per g fr wt of cells at day 9 withor without the addition of 25 µM potassium salicylate.More than 90% of the induced PR proteins was found in the mediumand less than 10% in the cells at day 9. Peroxidase activityin the medium was constant throughout the experiment althoughtotal activity in the flask increased with cell growth, indicatingthat PR proteins are actively secreted into the medium. (Received November 12, 1986; Accepted March 6, 1987)     

Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection

Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35−217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35−338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214−338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca²⁺ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM (N-terminal arginine-rich motif) appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.