Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

 We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. They were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P=0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients. Accepted: 29 April 1997     

Purification and characterization of eugenol dehydrogenase from Pseudomonas fluorescens E118

Pseudomonas fluorescens E118 was isolated from soil as an effective eugenol-degrading organism by a screening using eugenol as enrichment substrate. The first enzyme involved in the degradation of eugenol in this organism, eugenol dehydrogenase, was purified after induction by eugenol, and the purity of the enzyme was shown by SDS-PAGE and gel-permeation HLPC. The enzyme is a heterodimer that consists of a 10-kDa cytochrome c and a 58-kDa subunit. The larger subunit presumably contains flavin, suggesting a flavocytochrome c structure and an electron transfer via flavin and cytochrome c during dehydrogenation. The activity of the purified enzyme depended on the addition of a final electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, cytochrome c, or potassium ferricyanide. The enzyme catalyzed the dehydrogenation of three different 4-hydroxybenzylic structures including the conversion of eugenol to coniferyl alcohol, 4-alkylphenols to 1-(4-hydroxyphenyl)alcohols, and 4-hydroxybenzylalcohols to the corresponding aldehydes. The catalytic and structural similarity between this enzyme and a Penicillium vanillyl-alcohol oxidase and 4-alkylphenol methylhydroxylases from several Pseudomonas species is discussed. Received: 17 June 1998 / Accepted: 12 October 1998     

Structural Analysis ofArabidopsis thaliana Chromosome 5. VI. Sequence Features of the Regions of 1,367,185 bp Covered by 19 Physically Assigned P1 and TAC Clones

Nineteen Pl and TAC clones, which have been mapped on the finephysical map of the Arabidopsis thaliana chromosome 5, weresequenced according to the shotgun-based strategy, and theirstructural features were analysed. The total length of the regionssequenced in this study was 1,367,185 bp. Combining this withthe regions covered by 90 P1 and TAC clones proviously reported,the total length of chromosome 5 sequenced to date becomes 8,058,855bp. On the basis of similarity search against protein and ESTdatabases and gene modeling with computer programs, a totalof 330 potential protein-coding regions were identified, bringingan average density of the genes to approximately one gene per4.1 kb. Introns were identified in 81.0% of the potential proteingenes for which the entire gene structure was predicted, withan average number per gene of 4.2 and an average length of theintrons of 180 bp. The RNA-coding genes identified were 9 tRNAgenes corresponding to 8 amino acid species and 2 genes forU2 nuclear RNA. These sequence features are essentially identicalto those in the previously reported sequences. The sequencedata and gene information are available on the World Wide Webdatabase KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

Estradiol enhances binding to cultured human keratinocytes of antibodies specific for SS-A/Ro and SS-B/La. Another possible mechanism for estradiol influence of lupus erythematosus

A strong association between anti-SS-A/Ro and anti-SS-B/La antibodies and skin lesions has been well documented in subacute cutaneous lupus erythematosus and neonatal lupus erythematosis in which 70 to 80% of patients are female. In order to better understand the mechanisms of the influence of sex hormones on cutaneous lupus, we designed immunopathological in vitro experiments to evaluate the effects of estradiol and other sex steroids on the binding of SS-A/Ro- and SS-B/La-specific antibodies to cultured human keratinocytes from neonates. Cultured human keratinocytes incubated with antisera specific for SS-A/Ro or SS-B/La Ag were fixed with either acetone or paraformaldehyde and then analyzed in indirect immunofluorescent assays or by FACS analysis to detect cell surface IgG binding as an indirect measure of SS-A/Ro and SS-B/La Ag expression on the cell surface of keratinocytes. Estradiol (10(-5) to 10(-7) M) augmented binding of antiserum probes on the surface of cultured keratinocytes, with 10(-7) M estradiol showing the highest induction of cell surface binding of antisera specific for SS-A/Ro plus SS-B/La Ag (24.5% of cells were positive). In contrast, dihydrotestosterone, testosterone, and progesterone showed no augmentation. The augmentation by estradiol was partially inhibited by the antiestrogen nafoxidine. Estradiol augmented the relative incidence and absolute number of small or cuboidal cells binding antibodies specific for SS-A/Ro and SS-B/La Ag, whereas the number and incidence of larger differentiated cells binding anti-SS-A/Ro and anti-SS-B/La decreased significantly in cell cultures stimulated with estradiol. Flow cytometric analysis utilizing monospecific anti-SS-A/Ro or anti-SS-B/La sera showed that estradiol induced binding of anti-SS-A/Ro in 13.1% of cultured keratinocytes, of anti-SS-A/La in 14.4%, and of sera specific for both Ag in 21.4%. This direct association between estradiol and the augmentation of binding to the cell surface of human keratinocytes of IgG from antisera specific for SS-A/Ro and SS-B/La Ag may be a trigger factor of immunologic damage in lupus and may be important in the different sex rates observed in skin manifestation of subacute cutaneous and neonatal lupus erythematosis.     

An Experimental Test of Trade-Offs Associated with the Adaptation to Alternate Host Plants in the Introduced Herbivorous Beetle, <Emphasis Type="Italic">Ophraella communa</Emphasis>

The adaptation to alternate host plants of introduced herbivorous insects can be vital to agriculture due to the emergence of crop pests. Historically, it is assumed that there are trade-offs associated with the adaptation to new host plants; a generalist genotype that adapts to an alternate host is expected to have a relatively lower fitness on the ancestral host than a specialist genotype (physiological cost) or a relatively lower host-searching ability for the ancestral host plant (behavioral cost). In this study, we tested the costs of adaptation to a new host plant in the introduced herbivorous insect, Ophraella communa LeSage (Coleoptera: Chrysomelidae). In its native range (United States), O. communa feeds mostly on Ambrosia artemisiifolia L. (Asterales: Asteraceae) and cannot utilize the related species, Ambrosia trifida L. (Asterales: Asteraceae), as a host plant. On the other hand, the introduced O. communa population in Japan utilizes A. trifida extensively, and is adapting to it, both physiologically and behaviorally. We compared larval performance on the ancestral and alternate plants and adult host-searching ability between the native and introduced beetle populations. The introduced O. communa showed higher larval survival and adult feeding preference for the alternate host plant A. trifida than did the native O. communa, indicating that the introduced O. communa has rapidly adapted to the alternate host plant. However, there are no differences in either larval performance on the ancestral host A. artemisiifolia or host-searching accuracy between the native and introduced O. communa.     

Development of near-infrared firefly luciferin analogue reacted with wild-type and mutant luciferases

Interestingly, only the D-form of firefly luciferin produces light by luciferin–luciferase (L–L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L-form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L–L reaction, and the wavelengths of light in the near-infrared (NIR) range (700–900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L–L reactions with wild-type luciferase and/or mutant luciferase. Based on the structure–activity relationships, we designed and synthesized here a luciferin analogue with the 5-allyl-6-dimethylamino-2-naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild-type luciferase (λ_max = 705 nm) and mutant luciferase AlaLuc (λ_max = 655 nm).     

Effects of population density, sex morph, and tree size on reproduction in a heterodichogamous maple, Acer mono, in a temperate forest of Japan

The effects of local population density, sex morph [protogynous (PG) or protandrous (PA)], and individual tree size on the demographic processes of seed production were investigated in a heterodichogamous maple, Acer mono Maxim. var. Marmoratum (Nichols.) Hara f. dissectum, in a temperate forest of Japan. As the distance from conspecific reproductive adults increased, the percentage of immature seed fall and empty seeds increased significantly, indicating higher pollination success along with local population density. Although the difference was not distinct, pollination success was affected by the local population density of the reciprocal sex morph rather than that of both sex morphs. The trees at higher local population density sites suffered higher seed mortality due to predation and decay, and tended to produce smaller seeds. Thus, the impacts of local population density operated both positively and negatively on reproduction. As a factor of individual traits, tree size scarcely affected any demographic processes. On the other hand, sex morph did affect pollination success. Trees of PG type had lower immature seed fall than those of PA type, suggesting that the former has higher efficiency of pollen acceptance than the latter. The results on seed demography presented here partly support previous suggestions that heterodichogamous plants exhibit reciprocal cross-pollination and gender specialization as reproductive traits.     

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.     

Drosophila RecQ5 is involved in proper progression of early spermatogenesis

RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.