DNA-dependent adenosinetriphosphatase C1 from mouse FM3A cells has DNA helicase activity.

In our previous study, we identified four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, in mouse FM3A cells (Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529-533). The DNA-dependent ATPase C1 has been purified and characterized in detail. A divalent cation and a polynucleotide cofactor were required for the ATPase activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Almost no ATPase activity was observed with S1 nuclease-treated native DNA. ATPase C1 hydrolyzed ATP only among the ribo- and deoxyribonucleoside triphosphates tested, and this fact distinguished ATPase C1 from ATPases B, C2, and C3, because the latter enzymes are capable of hydrolyzing both ATP and dATP. The purified DNA-dependent ATPase C1 fraction was shown to have a DNA helicase activity that was dependent on hydrolysis of ATP. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.2 S on glycerol gradient centrifugation. Both activities showed similar preferences for nucleoside 5'-triphosphates and similar requirements for divalent cations. The DNA helicase activity was inhibited by the addition of single-stranded DNAs that served as cofactor for the ATPase activity. The efficiency of a single-stranded DNA to inhibit DNA helicase activity correlated well with the capacity of the DNA to serve as cofactor for DNA-dependent ATPase activity. The helicase was shown to migrate along the DNA strand in the 5' to 3' direction, which is the same direction of migration of the mouse DNA helicase B (Seki, M., Enomoto, T., Yanagisawa, J., Hanaoka, F., and Ui, M. (1988) Biochemistry 27, 1766-1771).     

Mechanism of Action and Development of Resistance to a New Amidino Penicillin

The mechanism of killing of Escherichia coli by a novel beta-lactam antibiotic, an amidino penicillin, has been investigated. This compound converts E. coli to relatively stable spherical forms at low concentration. However, the amidino penicillin caused no alteration in any of those parameters of peptidoglycan synthesis which can be studied. Above 10 mug of the antibiotic per ml the cells began to lyse, and a second mode of killing appeared. Mutants resistant to the amidino penicillin were isolated and several were studied in detail. Three mutant phenotypes were distinguished: (i) spherical shape and hypersensitive to lysis by either amidino penicillin or ampicillin; (ii) spherical shape and normally sensitive to lysis; (iii) rod shape, converted to viable spheres by amidino penicillin and normally sensitive to lysis.     

Characterization of a dissimilatory-type sulfite reductase, desulfoviridin, from Desulfovibrio africanus Benghazi

A desulfoviridin-type sulfite reductase having the alpha band at 638 nm was purified from Desulfovibrio africanus Benghazi (NCIB 8401) by chromatography on DEAE-cellulose, Sephadex G-200, and DEAE-Sepharose columns and by disc gel electrophoresis. The content of desulfoviridin in the soluble protein was estimated to be about 6% from the purification indexes. Like the typical desulfoviridin from D. vulgaris Miyazaki K, it formed mainly trithionate besides thiosulfate and sulfide in sulfite reduction coupled to hydrogenase and methyl viologen. No significant differences in the amino acid compositions, CD patterns in the UV (205-250 nm) region, and subunit structures were found, except for a pI value about 1 unit larger (pI 5.3). The split Soret (410 +/- 2 nm, less intense peak at 391 +/- 2 nm with a shoulder around 380 nm) and beta (584 +/- 2 nm) band maxima of the enzyme as isolated, and the visible absorption and fluorescence spectra of the acidic acetone-extracted chromophore were almost identical to those ascribed to sirohydrochlorin in spite of the reported difference in the native enzyme (alpha band maxima at 638 nm as against 628 +/- 2 nm in a typical desulfoviridin). Iron was the only significant chelatable metal contained in the chromophore. Some differences between africanus and vulgaris desulfoviridins were observed in the CD patterns in the UV to near UV region (250-340 nm) and also in the visible absorption spectra in the presence of dithionite.     

Expression and localization of diacylglycerol kinase isozymes and enzymatic features in rat lung

Diacylglycerol kinase (DGK) catalyzes phosphorylation of diacylglycerol to generate phosphatidic acid, and both molecules are known to serve as second messengers as well as important intermediates for the synthesis of various lipids. In this study, we investigated the spatiotemporal expression patterns of DGK isozymes together with the developmental changes of the mRNA expression and enzymatic property in rat lung. Northern blot and RT-PCR analyses showed that mRNAs for DGKalpha, -epsilon, and -zeta were detected in the lung. By immunohistochemical examination, DGKalpha and -zeta were shown to be coexpressed in alveolar type II cells and macrophages. Interestingly, these isozymes were localized at distinct subcellular locations, i.e., DGKalpha in the cytoplasm and DGKzeta in the nucleus, suggesting different roles for these isozymes. In the developing lung, the expression for DGKalpha and -zeta was transiently elevated on embryonic day 21 (E21) to levels approximately two- to threefold higher than on postnatal day 0 (P0). On the other hand, the expression for DGKepsilon was inversely elevated approximately twofold on P0 compared with that on E21. These unique changes in the expression pattern during the perinatal period suggest that each isozyme may play a distinct role in the adaptation of the lung to air or oxygen breathing at birth.     

Crystal structure of the N-terminal RecA-like domain of a DEAD-box RNA helicase, the Dugesia japonica vasa-like gene B protein

The Dugesia japonica vasa-like gene B (DjVLGB) protein is a DEAD-box RNA helicase of a planarian, which is well known for its strong regenerative capacity. DjVLGB shares sequence similarity to the Drosophila germ-line-specific DEAD-box RNA helicase Vasa, and even higher similarity to its paralogue, mouse PL10. In this study, we solved the crystal structure of the DjVLGB N-terminal RecA-like domain. The overall fold and the structures of the putative ATPase active site of the DjVLGB N-terminal RecA-like domain are similar to those of the previously reported DEAD-box RNA helicase structures. In contrast, the surface structure of the side opposite to the putative ATPase active site is different from those of the other DEAD-box RNA helicases; the characteristic hydrophobic pockets are formed with aromatic and proline residues. These pocket-forming residues are conserved in the PL10-subfamily proteins, but less conserved in the Vasa orthologues and not conserved in the DEAD-box RNA helicases. Therefore, the structural features that we found are characteristic of the PL10-subfamily proteins and might contribute to their biological roles in germ-line development.     

Effect of dietary anti-urease immunoglobulin Y on Helicobacter pylori infection in Mongolian gerbils

BACKGROUND AND AIM: Helicobacter pylori is known to be a major pathogenic factor in the development of gastritis, peptic ulcer disease and gastric cancer. Recently, chicken egg yolk immunoglobulin Y (IgY) has been recognized as an inexpensive antibody source for passive immunization against gastrointestinal infections. The present study was designed to investigate the effect of anti-urease IgY on H. pylori infection in Mongolian gerbils. METHODS: H. pylori-infected Mongolian gerbils were administered a diet containing anti-urease IgY, with or without famotidine (F). After 10 weeks, bacterial culture and measurement of the gastric mucosal myeloperoxidase (MPO) activity were performed. In a second experiment, another group of gerbils was started on a diet containing F + IgY a week prior to H. pylori inoculation. After 9 weeks, these animals were examined. RESULTS: In the H. pylori-infected gerbils, there were no significant differences in the level of H. pylori colonization among the different dietary and control groups. However, the MPO activity was significantly decreased in the H. pylori group administered the F + IgY diet compared with that in the H. pylori group administered the IgY, F, or control diet. Furthermore, in the gerbils administered the F + IgY diet prior to the bacterial inoculation, inhibition of H. pylori colonization and suppression of the elevated gastric mucosal MPO activity were observed. CONCLUSIONS: Oral administration of urease-specific IgY not only inhibited H. pylori disease activity in H. pylori-infected gerbils, but also prevented H. pylori colonization in those not yet infected. These encouraging results may pave the way for a novel therapeutic and prophylactic approach in the management of H. pylori-associated gastroduodenal disease.     

Comprehensive Structural Analysis of the Genome of Red Clover (Trifolium pratense L.)

With the aim of establishing the basic knowledge and resourcesneeded for applied genetics, we investigated the genome structureof red clover Trifolium pratense L. by a combination of cytological,genomic and genetic approaches. The deduced genome size was440 Mb, as estimated by measuring the nuclear DNA content byflow cytometry. Seven chromosomes could be distinguished bymicroscopic observation of DAPI stained prometaphase chromosomesand fluorescence in situ hybridization using 28S and 5S rDNAprobes and bacterial artificial chromosome probes containingmicrosatellite markers with known positions on a genetic linkagemap. The average GC content of the genomes of chloroplast, mitochondrionand nucleus were shown to be 33.8, 42.9 and 34.2%, respectively,by the analysis of 1.4 Mb of random genomic sequences. A totalof 26 356 expressed sequence tags (ESTs) that were grouped into9339 non-redundant sequences were collected, and 78% of theESTs showed sequence similarity to registered genes, mainlyof Arabidopsis thaliana and rice. To facilitate basic and appliedgenetics in red clover, we generated a high-density geneticlinkage map with gene-associated microsatellite markers. A totalof 7159 primer pairs were designed to amplify simple sequencerepeats (SSRs) identified in four different types of libraries.Based on sequence similarity, 82% of the SSRs were likely tobe associated with genes. Polymorphism was examined using twoparent plants, HR and R130, and 10 F₁ progeny by agarose gelelectrophoresis, followed by genotyping for the primer pairsshowing polymorphisms using 188 F₁ plants from the mapping population.The selected 1305 microsatellite markers as well as the previouslydeveloped 167 restriction fragment length polymorphism markerswere subjected to linkage analysis. A total of 1434 loci detectedby 1399 markers were successfully mapped onto seven linkagegroups totaling 868.7 cM in length; 405 loci (28%) were bi-parental,611 (43%) were specific to HR and 418 (29%) were specific toR130. Each genetic linkage group was linked to a correspondingchromosome by FISH analysis using seven microsatellite markersspecific to each of the linkage groups as probes. Transferabilityof the developed microsatellite markers to other germplasmswas confirmed by testing 268 selected markers on 88 red clovergermplasms. Macrosynteny at the segmental level was observedbetween the genomes of red clover and two model legumes, Lotusjaponicus and Medicago truncatula, strongly suggesting thatthe genome information for the model legumes is transferableto red clover for genetic investigations and experimental breeding.     

Loss of integrin α3 expression is associated with acquisition of invasive potential by ovarian clear cell adenocarcinoma cells

Among various types of surface epithelial ovarian carcinoma, clear cell adenocarcinoma often has a particularly poor prognosis even when diagnosed in stage I. It is resistant to existing anticancer drugs and appears to have different biological properties to other histological types of ovarian cancer. The present study was conducted using cell lines derived from ovarian clear cell adenocarcinoma in order to identify genes associated with the acquisition of malignant potential by this type of cancer. Two cell lines derived from ovarian clear cell adenocarcinoma (RMG-I and RMG-V), with different levels of invasive potential in an invasion assay, were used. DNA fragments were extracted from the band showing differences in the level of expression by RT-PCR with fluorescent differential display. A total of 56 different DNA base sequences were determined by direct sequencing. Primers were established using these base sequences and the level of expression in each cancer cell line was determined by real-time PCR. Integrin a3, the gene of which is present on chromosome 17q, was identified. It was also detected by a microarray analysis as one of the molecules showing a different level of expression between the two cell lines. Then the pattern of integrin a3 expression was investigated using immunocytochemical staining, and was found to differ between the two cell lines. The findings obtained using these cell lines derived from ovarian clear cell adenocarcinoma indicate that integrin alpha3 may associated with the acquisition of malignant potential by clear cell adenocarcinoma.