Takashi Umeda

Specific Interactions between the Ferredoxin and Terminal Oxygenase Components of a Class IIB Rieske Nonheme Iron Oxygenase, Carbazole 1,9a-Dioxygenase

Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (Oxy), ferredoxin (Fd), and ferredoxin reductase (Red) components and is a member of the Rieske nonheme iron oxygenases. Rieske nonheme iron oxygenases are divided into five subclasses (IA, IB, IIA, IIB, and III) based on the number of constituents and the nature of their redox centers. Each component of a class IIB CARDO from Nocardioides aromaticivorans IC177 was purified, and the interchangeability of the electron transfer reactions with each component from the class III CARDOs was investigated. Despite the fact that the Fds of both classes are Rieske-type, strict specificities between the Oxy and Fd components were observed. On the other hand, the Fd and Red components were interchangeable, even though the Red components differ in cofactor composition; the class IIB Red contains flavin-adenine-dinucleotide (FAD)- and NADH-binding domains, whereas the class III Red has a chloroplast-type [2Fe-2S] cluster in addition to the FAD- and NADH-binding domains. The crystal structures of the class IIB Oxy and Fd components were compared to the previously reported Fd:Oxy complex structure of class III CARDO. This comparison suggested residues in common between class IIB and class III CARDOs that are important for interactions between Fd and Oxy. In the class IIB CARDOs, these included His75 and Glu71 in Fd and Lys20 and Glu357 in Oxy for electrostatic interactions, and Phe74 and Pro90 in Fd and Trp21, Leu359, and Val367 in Oxy for hydrophobic interactions. The residues that formed the interacting surface but were not conserved between classes were thought to be necessary to form the appropriate geometry and to determine electron transfer specificity between Fd and Oxy.

Nuclear magnetic resonance imaging of a benign cystic teratoma in the retroperitoneum.

A retroperitoneal teratoma arising from the vicinity of the upper pole of the right kidney in an 18-year-old woman is described. The plain anteroposterior abdominal radiographs showed irregular calcific shadows with tooth-like calcifications within the right upper quadrant of the abdomen. The findings of a primary cystic teratoma in the retroperitoneum examined by computerized tomography and magnetic resonance imaging of the abdomen are presented. Sagittal and coronal magnetic resonance imaging scans were useful to determine the extent of the mass, and to distinguish the mass from adjacent structures and organs.

Intravesical treatment of bladder cancer with recombinant human interferon-β : intravesical GKT-β chemotherapy research group (Chairman : Tadao Niijima)

SummaryIn order to examine its clinical efficacy, recombinant human interferon-β (rIFN-β) was instilled intravesically into 51 patients with superficial bladder cancer. Ten patients, who received intermittent intravesical instillation at a dose of (3−36) × 106 U rIFN-β on days 1–3 every week, showed no response. Thirty-two patients received intravesical instillation at a dose of (3−36) × 106 U every day for 10–20 days. Eight patients showed partial response, indicating an efficacy rate of 25%. Nine patients received divided doses of 18 × 106 U twice a day every day for 10–20 days. Six patients showed partial response, indicating an efficacy rate of 67%. This value was significantly higher than that obtained by administering divided doses. The response to intravesical instillation therapy with rIFN-β varies with treatment protocol. Frequent and longer exposure to rIFN-β may induce better regression of superficial bladder cancer. Six incidences of side-effects were found in five cases (9.8%): pollakiuria in one, pain on micturition in two, fever in two, and eruption in one case. All of these side-effects were slight and reversible after drug withdrawal. Laboratory tests showed only a few changes with low severity. Thus, rIFN-β is potentially a new drug for instillation therapy of superficial bladder cancer, in view of the absence of adverse effects.

Detection of Fc-receptor on human renal glomerulus.

Abstract Six surgically removed normal human kidney specimens were examined by rosette formation techniques on tissue sections. Sheep red cells (SRC) sensitized with IgG hemolysin (IgG-EA) and those with IgM hemolysin and complement (IgM-EAC) exclusively stuck to renal glomeruli, while SRC sensitized with reduced and alkylated IgG hemolysin ( R A IgG-EA) and those with IgM hemolysin only (IgM-EA) or nonsensitized SRC (E) did not stick to renal specimens. The sticking of IgG-EA was significantly inhibited by aggregated human IgG, as well as by the sera from patients with active SLE. In the present study, the evidence that human renal glomeruli bear not only complement receptors but also Fc-receptors was presented. The possibility was discussed that circulating immune complexes may be trapped into renal glomeruli by these receptors and will result in the depositions as observed in immune complex nephritis such as lupus nephritis.

Oligomerization and DNA-binding capacity of Pmr, a histone-like protein H1 (H-NS) family protein encoded on IncP-7 carbazole-degradative plasmid pCAR1.

Pmr, a histone-like protein H1 (H-NS) family protein encoded on plasmid pCAR1, is a key factor in optimizing gene transcription on both pCAR1 and the host chromosome. To clarify the mode of function of Pmr, we performed gel filtration chromatography analysis and protein-protein cross-linking, and found that Pmr forms homo-oligomers, consisting of its homodimers. We also found, by atomic force microscopy, that Pmr has DNA-bridging capacity. From these results, Pmr was deduced to have features common to H-NS family proteins. Additionally, evaluating protein-DNA affinity is important to clarify the mode of function of Pmr, and hence we performed an electrophoretic mobility shift assay. Though Pmr formed high-order protein-DNA complexes and did not show preference for nucleic acid sequences, the C-terminal region of Pmr did, suggesting that the DNA-binding affinity of Pmr can be evaluated by using its C-terminal region.

Surface Markers of Lymphocytes Infiltrating Seminoma Tissue

Infiltrating lymphocytes were separated from human seminoma tissue obtained surgically. The relative distributions of B and T lymphocytes, and a subset of T lymphocytes with IgG-Fc receptors were measured by the rosette-forming method in peripheral blood and in seminoma tissue from 7 patients with pure seminoma of the testis. The proportions of T and TG cells were increased in seminoma tissue compared to those of peripheral blood from the same patients, whereas the proportion of B cells did not change. This result might correlate with the favorable prognosis of seminoma usually accompanied with lymphocytic infiltration.

Alteration of the Substrate Specificity of the Angular Dioxygenase Carbazole 1,9a-Dioxygenase

Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (CARDO-O) and electron transport components. CARDO can catalyze specific oxygenation for various substrates: angular dioxygenation for carbazole and dibenzo-p-dioxin, lateral dioxygenation for anthracene, and monooxygenation for methylene carbon of fluorene and sulfide sulfur of dibenzothiophene. To elucidate the molecular mechanism determining its unique substrate specificity, 17 CARDO-O site-directed mutants at amino acid residues I262, F275, Q282, and F329, which form the substrate-interacting wall around the iron active site by CARDO-O crystal structure, were generated and characterized. F329 replacement dramatically reduced oxygenation activity. However, several mutants produced different products from the wild-type enzyme to a large extent: I262V and Q282Y (1-hydroxycarbazole), F275W (4-hydroxyfluorene), F275A (unidentified cis-dihydrodiol of fluoranthene), and I262A and I262W (monohydroxydibenzothiophenes). These results suggest the possibility that the respective substrates bind to the active sites of CARDO-O mutants in a different orientation from that of the wild-type enzyme.

Crystallization and preliminary X-ray diffraction studies of a ferredoxin reductase component of carbazole 1,9a-dioxygenase from Novosphingobium sp. KA1.

Carbazole 1,9a-dioxygenase (CARDO) is the initial enzyme of the carbazole-degradation pathway. The CARDO of Novosphingobium sp. KA1 consists of a terminal oxygenase, a putidaredoxin-type ferredoxin and a ferredoxin-NADH oxidoreductase (Red) and is classified as a class IIA Rieske oxygenase. Red from KA1 was crystallized at 278 K by the hanging-drop vapour-diffusion method using PEG 4000. The crystal diffracted to 1.58 A resolution and belonged to space group P3(2), with unit-cell parameters a = b = 92.2, c = 78.6 A, alpha = gamma = 90, beta = 120 degrees . Preliminary analysis of the X-ray diffraction data revealed that the asymmetric unit contained two Red monomers. The crystal appeared to be a merohedral twin, with a twin fraction of 0.32 and twin law (-h, -k, l).

Crystallization and preliminary X-ray diffraction studies of a terminal oxygenase of carbazole 1,9a-dioxygenase from Novosphingobium sp. KA1

Carbazole 1,9a-dioxygenase (CARDO) is the initial dioxygenase in the carbazole-degradation pathway of Novosphingobium sp. KA1. The CARDO from KA1 consists of a terminal oxygenase (Oxy), a putidaredoxin-type ferredoxin and a ferredoxin reductase. The Oxy from Novosphingobium sp. KA1 was crystallized at 277u2005K using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. Diffraction data were collected to a resolution of 2.1u2005Å. The crystals belonged to the monoclinic space group P2(1). Self-rotation function analysis suggested that the asymmetric unit contained two Oxy trimers; the Matthews coefficient and solvent content were calculated to be 5.9u2005Å(3)u2005Da(-1) and 79.1%, respectively.

Crystallization and preliminary X-ray diffraction studies of a novel ferredoxin involved in the dioxygenation of carbazole by Novosphingobium sp. KA1

Novosphingobium sp. KA1 uses carbazole 1,9a-dioxygenase (CARDO) as the first dioxygenase in its carbazole-degradation pathway. The CARDO of KA1 contains a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. In contrast to the CARDO systems of other species, the ferredoxin component of KA1 is a putidaredoxin-type protein. This novel ferredoxin was crystallized at 293 K by the hanging-drop vapour-diffusion method using PEG MME 550 as the precipitant under anaerobic conditions. The crystals belong to space group C222(1) and diffraction data were collected to a resolution of 1.9 A (the diffraction limit was 1.6 A).