Identification of the transgenic integration site in immunodeficient tgε26 human CD3ε transgenic mice

A strain of human CD3ε transgenic mice, tgε26, exhibits severe immunodeficiency associated with early arrest of T cell development. Complete loss of T cells is observed in homozygous tgε26 mice, but not in heterozygotes, suggesting that genomic disruption due to transgenic integration may contribute to the arrest of T cell development. Here we report the identification of the transgenic integration site in tgε26 mice. We found that multiple copies of the human CD3ε transgene are inserted between the Sstr5 and Metrn loci on chromosome 17, and that this is accompanied by duplication of the neighboring genomic region spanning 323 kb. However, none of the genes in this region were abrogated. These results suggest that the severe immunodeficiency seen in tgε26 mice is not due to gene disruption resulting from transgenic integration.     

X-ray Structures of Human Galectin-9 C-terminal Domain in Complexes with a Biantennary Oligosaccharide and Sialyllactose

Galectin-9, a tandem-repeat-type β-galactoside-specific animal lectin with two carbohydrate recognition domains (CRDs) at the N- and C-terminal ends, is involved in chemoattraction, apoptosis, and the regulation of cell differentiation and has anti-allergic effects. Its ability to recognize carbohydrates is essential for its biological functions. Human galectin-9 (hG9) has high affinity for branched N-glycan-type oligosaccharides (dissociation constants of 0.16–0.70 μm) and linear β1–3-linked poly-N-acetyllactosamines (0.09–8.3 μm) and significant affinity for the α2–3-sialylated oligosaccharides (17–34 μm). Further, its N-terminal CRD (hG9N) and C-terminal CRD (hG9C) differ in specificity. To elucidate this unique feature of hG9, x-ray structures of hG9C in the free form and in complexes with N-acetyllactosamine, the biantennary pyridylaminated oligosaccharide, and α2–3-sialyllactose were determined. They are the first x-ray structural analysis of C-terminal CRD of the tandem-repeat-type galectin. The results clearly revealed the mechanism by which branched and α2–3-sialylated oligosaccharides are recognized and explained the difference in specificity between hG9N and hG9C. Based on structural comparisons with other galectins, we propose that the wide entrance for ligand binding and the shallow binding site of hG9C are favorable for branched oligosaccharides and that Arg²²¹ is responsible for recognizing sialylated oligosaccharides.     

Structure-activity relationship of uniconazole, a potent inhibitor of ABA 8'-hydroxylase, with a focus on hydrophilic functional groups and conformation

The plant growth retardant S-(+)-uniconazole (UNI-OH) is a strong inhibitor of abscisic acid (ABA) 8'-hydroxylase, a key enzyme in the catabolism of ABA, a plant hormone involved in stress tolerance, stomatal closure, flowering, seed dormancy, and other physiological events. In the present study, we focused on the two polar sites of UNI-OH and synthesized 3- and 2'-modified analogs. Conformational analysis and an in vitro enzyme inhibition assay yielded new findings on the structure-activity relationship of UNI-OH: (1) by substituting imidazole for triazole, which increases affinity to heme iron, we identified a more potent compound, IMI-OH; (2) the polar group at the 3-position increases affinity for the active site by electrostatic or hydrogen-bonding interactions; (3) the conformer preference for a polar environment partially contributes to affinity for the active site. These findings should be useful for designing potent azole-containing specific inhibitors of ABA 8'-hydroxylase.     

Lipoprotein from the outer membrane of Escherichia coli: purification, paracrystallization, and some properties of its free form.

In the envelope of Escherichia coli, is a lipoprotein of molecular weight 7,200 as a major envelope protein. This lipoprotein was previously shown to exist in two different forms in the outer membrane of E. coli: the free form and the boundform, which is covalently linked to the peptidoglycau. The free form of the lipoprotein has been purified and paracrystallized by adding acetone to a sodium dodecyl sulfate solution in the presence of magnesium ion. The paracrystals were needle shaped. An electron micrograph of the negatively stained paracrystals showed a highly ordered ultrastructure. The chemical structure of the free form was compared with that of the bound form by (i) the amino acid composition, (ii) the fatty acid composition, and (iii) the peptide analysis after cyanogen bromide cleavage. The alpha-helical content of the free form of the lipoprotein was measured from the circular dichroism spectrum of the lipoprotein in 0.01% sodium dodecyl sulfate and found to be 87%. Using the purified lipoprotein as antigen, antiserum against the free form of the lipoprotein was obtained. Immunoprecipitation of the lipoprotein with the antiserum was found to be very specific, since only the free form of the lipoprotein was found as a major peak when the antiserum was reacted with the whole envelope proteins solubilized in 0.2% sodium dodecyl sulfate, and the immunoprecipitate thus formed was analyzed by polyacrylamide gel electrophoresis.     

Cloning and Characterization of a Gene Cluster for Hatomarubigin Biosynthesis in Streptomyces sp. Strain 2238-SVT4

Streptomyces sp. strain 2238-SVT4 produces hatomarubigins A, B, C, and D, which belong to the angucycline family. Among them, hatomarubigin D has a unique dimeric structure with a methylene linkage. PCR using aromatase and cyclase gene-specific primers identified the hrb gene cluster for angucycline biosynthesis in Streptomyces sp. 2238-SVT4. The cluster consisted of 30 open reading frames, including those for the minimal polyketide synthase, ketoreductase, aromatase, cyclase, O-methyltransferase, oxidoreductase, and oxygenase genes. Expression of a part of the gene cluster containing hrbR1 to hrbX in Streptomyces lividans TK23 resulted in the production of hatomarubigins A, B, and C. Hatomarubigin D was obtained from the conversion of hatomarubigin C by a purified enzyme encoded by hrbY, among the remaining genes.The angucycline antibiotics are a large group of naturally occurring aromatic polyketides of microbial origin (11, 15). They exhibit a wide range of biological activities, which include antibacterial, antiviral, antitumor, enzyme inhibitory, and platelet aggregation inhibitory effects. Although all the members contain a benz[a]anthraquinone skeleton of decaketide origin, their structural diversity is very broad and they have a wide variety of oxidation states. Hatomarubigins A, B, C, and D (Fig. ​(Fig.1)1) belong to the angucycline family and reverse colchicine resistance in multidrug-resistant tumor cells (8). Among them, hatomarubigin D is a unique hatomarubigin C dimer with a methylene linkage. Such a dimer has not been reported previously, and little is known about the mechanism of the methylene bridge formation between two aromatic rings. In this study, a gene cluster for hatomarubigin biosynthesis was identified in Streptomyces sp. strain 2238-SVT4, and a part of the gene cluster was expressed in Streptomyces lividans to produce the hatomarubigins.Open in a separate windowFIG. 1.Structures of angucycline antibiotics.     

The Arabidopsis-accelerated cell death gene ACD1 is involved in oxygenation of pheophorbide a: inhibition of the pheophorbide a oxygenase activity does not lead to the "stay-green" phenotype in Arabidopsis

Oxygenation of pheophorbide a is a key step in chlorophyll breakdown. Several biochemical studies have implicated that this step was catalyzed by an iron-containing and ferredoxin-dependent monooxygenase, pheophorbide a oxygenase (PaO). It has been proposed that inhibition of its activity arrests the chlorophyll breakdown and leads to the "stay-green" phenotype. We searched the Arabidopsis genome for a possible PaO-encoding gene and hypothesized that it has homology to known iron-containing Rieske-type monooxygenase sequences. We identified three such open reading frames, Tic55, ACD1 and ACD1-like. We produced transgenic Arabidopsis plants which expressed antisense RNA as a method to inhibit the expression of these genes. The appearance of these antisense plants were indistinguishable from that of the wild type under illumination. However, after they were kept under darkness for 5 d and again illuminated, the leaves of the antisense ACD1 plants (AsACD1) were bleached. Leaves of AsACD1 accumulated 387 nmol (g FW)(-1) pheophorbide a which corresponded to 60% of chlorophyll a degraded. The rate of decrease in chlorophyll a was not influenced in senesced AsACD1 leaves. These results demonstrated that ACD1 is involved in PaO activity, and its inhibition led to photooxidative destruction of the cell instead of the "stay-green" phenotype.