Purification and Characterization of Thin Pili of IncI1 Plasmids ColIb-P9 and R64: Formation of PilV-Specific Cell Aggregates by Type IV Pili

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.     

Assessment of Poliovirus Eradication in Japan: Genomic Analysis of Polioviruses Isolated from River Water and Sewage in Toyama Prefecture

Seventy-eight poliovirus strains isolated from river water and sewage in Toyama Prefecture, Japan, during 1993 to 1995 were characterized by the PCR-restriction fragment length polymorphism (RFLP) method and by partially sequencing the VP3 and VP1 regions of the viral genome. Of these isolates, 36 were identified as Sabin vaccine strains, and 42 were identified as vaccine variant strains that had less than 1.4% nucleotide divergence from the Sabin strains, including 7 isolates with patterns different from those of Sabin strains as determined by PCR-RFLP analysis. These findings suggest that wild-type poliovirus was not circulating in Toyama Prefecture.     

Involvement of Moc1 in sexual development and survival of Schizosaccharomyces pombe

The moc1 gene in Schizosaccharomyces pombe was found as to overcome sterility caused by high expression of adenylyl cyclase. The moc1 gene was found to be identical with sds23 and psp1. Although psp1 has been reported to be essential for growth, sds23 has not been. To clarify this apparent discrepancy, we first assessed independently the phenotypes of the moc1 disruptant. We confirmed that the deletion mutant of moc1 is sterile, sensitive to high salt, and grows slowly at higher and lower temperatures, and that mutant cells are elongated. Besides these phenotypes, we found that viability of the moc1 disruptant was rapidly lost at the stationary phase. We confirmed that the Moc1 protein is phosphorylated in the stationary phase and also under nitrogen-starved conditions. We examined the significance of this phosphorylation of Moc1 by creating the S333A or S333D mutant Moc1. Interestingly, while S333D mutant Moc1 is lower in inducing sexual development, S333A mutant Moc1 is higher in this than the wild type, suggesting that phosphorylation of Moc1 affects sexual development. The other phenotypes, such as sensitivity to high salt and higher temperature and elongation of cells, were not affected by mutation of S333A nor S333D. We found that Moc1-GFP localized to both the cytosol and the nucleus during mitotic growth, but accumulated in the nucleus in mating cells and then enriched in spores, and that this localization shift was negatively regulated by the cAMP pathway. This and the observations above suggest that nuclear localized Moc1 is an inducer of sexual development. Thus, in addition to the roles of moc1/sds23/psp1 in mitosis and stress response, it is also important for the survival and sexual development of fission yeast, but phosphorylation of Moc1 only affects the sexual development.     

Cytologic features of functioning stromal cells in a yolk sac tumor of the ovary. A case report

BACKGROUND: Functioning stromal cells are sometimes seen in primary and metastatic ovarian neoplasms. However, the cytologic features of functioning stromal cells have been described only rarely. CASE: A 19-year-old woman had an alpha-fetoprotein-producing ovarian yolk sac tumor with functioning stroma. Her preoperative serum testosterone level was elevated. Imprint cytology showed that the functioning stromal cells had centrally located nuclei with low nuclear/cytoplasmic ratios. Occasionally these cells had vacuolated cytoplasm, suggesting the presence of lipids. In sharp contrast, the yolk sac tumor cells had more pleomorphic and hyperchromatic nuclei. We were able to distinguish between neoplastic and functioning stromal cells on the basis of these findings. In addition, immunostaining for inhibin on imprint cytologic slides was of great help in identifying functioning stromal cells. CONCLUSION: Because functioning stromal cells may unexpectedly induce hormonal effects in a variety of ovarian tumors, it is important to identify such cells in cytologic specimens.     

Phylogenetic analysis of spotted fever group rickettsiae based on gltA, 17-kDa, and rOmpA genes amplified by nested PCR from ticks in Japan

In order to understand the natural situation of rickettsiae in the ticks in Japan, the rickettsial genes, gltA gene, rOmpA gene, and 17-kDa gene, were amplified from the ticks by nested PCR. The prevalences of rickettsial gltA genes among Haemaphysalis formosensis, H. longicornis, H. megaspinosa, Ixodes ovatus, H. flava, H. kitaokai, and I. persulcatus were 62, 57, 24, 24, 19, 13, and 10%, respectively; 26% (186/722) being the average. The gltA genes amplified from the ticks were classified into 9 genotypes (I to IX) by the difference in nucleotide sequences. Genotype I was detected from 7 species of ticks. Genotype II mainly was detected from H. longicornis and H. formosensis. Genotypes III and VII mainly were detected from H. flava and I. ovatus. The polarization in the distribution of genotypes among regions where the ticks were collected was not clear. Based on the phylogenetic analysis of the three genes presented here, genotypes I, III, and IV (detected from H. formosensis, H. hystricia, and I. ovatus ) are genetically close with each other, but rickettsiae of the same property still have not been isolated from ticks anywhere in the world. These genotypes should be considered as new species among SFG rickettsiae. Genotype II was identical with strain FUJ-98, genetically close to R. japonica which has been isolated from ticks in China. Genotype V was identical with R. felis and strain California 2 isolated from the cat flea. This is the first report on the detection of R. felis from ticks. Genotype VI detected from Ixodes sp. did not seem to belong to genus Rickettsia. Based on the previous antigenic data and the phylogenetic analysis presented here, Genotype VII should be considered a variant of R. helvetica and genotype VIII detected from I. ovatus and I. persulcatus were identical with R. helvetica. Genotype IX detected from I. nipponensis was genetically close to the strains IRS3, IRS4, and IrR/Munich isolated from I. ricinus in Slovakia and German.     

Crystal structure of human L-xylulose reductase holoenzyme: probing the role of Asn107 with site-directed mutagenesis

L-Xylulose reductase (XR), an enzyme in the uronate cycle of glucose metabolism, belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. Among the SDR enzymes, XR shows the highest sequence identity (67%) with mouse lung carbonyl reductase (MLCR), but the two enzymes show different substrate specificities. The crystal structure of human XR in complex with reduced nicotinamide adenine dinucleotide phosphate (NADPH) was determined at 1.96 A resolution by using the molecular replacement method and the structure of MLCR as the search model. Features unique to human XR include electrostatic interactions between the N-terminal residues of subunits related by the P-axis, termed according to SDR convention, and an interaction between the hydroxy group of Ser185 and the pyrophosphate of NADPH. Furthermore, identification of the residues lining the active site of XR (Cys138, Val143, His146, Trp191, and Met200) together with a model structure of XR in complex with L-xylulose, revealed structural differences with other members of the SDR family, which may account for the distinct substrate specificity of XR. The residues comprising a recently proposed catalytic tetrad in the SDR enzymes are conserved in human XR (Asn107, Ser136, Tyr149, and Lys153). To examine the role of Asn107 in the catalytic mechanism of human XR, mutant forms (N107D and N107L) were prepared. The two mutations increased K(m) for the substrate (>26-fold) and K(d) for NADPH (95-fold), but only the N107L mutation significantly decreased k(cat) value. These results suggest that Asn107 plays a critical role in coenzyme binding rather than in the catalytic mechanism.     

Direct measure of functional importance visualized atom-by-atom for photoactive yellow protein: application to photoisomerization reaction

Photoreceptor proteins serve as efficient nano-machines for the photoenergy conversion and the photosignal transduction of living organisms. For instance, the photoactive yellow protein derived from a halophilic bacterium has the p-coumaric acid chromophore, which undergoes an ultrafast photoisomerization reaction after light illumination. To understand the structure-function relationship at the atomic level, we used a computational method to find functionally important atoms for the photoisomerization reaction of the photoactive yellow protein. In the present study, a "direct" measure of the functional significance was quantitatively evaluated for each atom by calculating the partial atomic driving force for the photoisomerization reaction. As a result, we revealed the reaction mechanism in which the specific role of each functionally important atom has been well characterized in a systematic manner. In addition, we observed that this mechanism is strongly conserved during the thermal fluctuation of the photoactive yellow protein. We compared the experimental data of fluorescence decay constant of several different mutants and the present analysis. As a result, we found that the reaction rate constant is decreased when a large positive driving force is missing.     

Characterization of a major form of human isatin reductase and the reduced metabolite.

Isatin, an endogenous indole, has been shown to inhibit monoamine oxidase, and exhibit various pharmacological actions. However, the metabolism of isatin in humans remains unknown. We have found high isatin reductase activity in the 105,000 g supernatants of human liver and kidney homogenates, and have purified and characterized a major form of the enzyme in the two tissues. The hepatic and renal enzymes showed the same properties, including an M(r) of 31 kDa, substrate specificity for carbonyl compounds and inhibitor sensitivity, which were also identical to those of recombinant human carbonyl reductase. The identity of the isatin reductase with carbonyl reductase was immunologically demonstrated with an antibody against the recombinant carbonyl reductase. About 90% of the soluble isatin reductase activity in the liver and kidney was immunoprecipitated by the antibody. The Km (10 microm) and k(cat)/K(m) (1.7 s(-1) x microm(-1)) values for isatin at pH 7.0 were comparable to those for phenanthrenequinone, the best xenobiotic substrate of carbonyl reductase. The reduced product of isatin was chemically identified with 3-hydroxy-2-oxoindole, which is also excreted in human urine. The inhibitory potency of the reduced product for monoamine oxidase A and B was significantly lower than that of isatin. The results indicate that the novel metabolic pathway of isatin in humans is mediated mainly by carbonyl reductase, which may play a critical role in controlling the biological activity of isatin.