Hematoporphyrin/DAPI staining: simplified simultaneous one-step staining of DNA and cell protein and trial application in automated cytological screening by flow cytometry

We describe a procedure for simplified, simultaneous one-step staining in 10 min for DNA and cell and tissue proteins using a newly developed staining solution containing 0.03% hematoporphyrin (HP) with 0.001% DAPI [or with Hoeschst 33342 (HO)]. These HP/DAPI or HP/HO solutions were especially developed to facilitate a trial of automated cancer cell screening on sputum samples using flow cytometry. Under UV light (365 nm) with fluorescence microscopy, HP/DAPI-stained cells showed red fluorescence (max. 670 nm) of cytoplasm and simultaneous blue fluorescence (max. 470 nm) of nuclei. The distance between the maximum peak of fluorescence spectra of DNA and that of protein was as large as 200 nm, and there was no detectable overlapping of each spectrum at the photometric filter range, which provided accurate measurement of DNA and protein. On flow cytometry, a single UV beam (370 nm) from the argon laser was used for excitation of both dyes. Measurement of DNA was done using a 470-nm bandpass filter and of protein using a 640-nm longpass (or 670-nm bandpass) filter. Reflecting the undetectable overlapping of the fluorescence spectra of protein and DNA, normal diploid cells in sputum revealed horizontal distributions along the 2C level on the dot-plot display of flow cytometry, which made sorting of abnormal hyperdiploid cells and cancer cells easier.     

Characterization of pathogenic constituents of Cryptococcus neoformans strains

We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 X 10(5) or 1 X 10(7) viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; a cell surface structure for responsible for resistance to intra- or extracellular killing and induction of the granulomatous lesion; a growth rate suitable for interacting with phagocytic elimination.     

Differential Regulation of Intracellular Signaling Systems by Sodium Fluoride in Rat Glioma Cells

Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca²⁺ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca²⁺-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 µ M BAPTA tetraacetoxymethyl ester or in the presence of W-7 in a concentration-dependent manner. N ^G-Monomethyl- l -arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca²⁺]_i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca²⁺]_i was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca²⁺ was reduced by pretreating the cells with NaF. The reduction in Ca²⁺ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca²⁺]_i, and 5-HT_2A receptor function in C6 glioma cells.     

Murine defense mechanism against Candida albicans infection. I. Collaboration of cell-mediated and humoral immunities in protection against systemic C. albicans infection

Mice immunized with viable C. albicans cells demonstrated a high incidence of cell-mediated and a low incidence of humoral immune response. There was good agreement between the final survival rate of C. albicans infected mice and the rate of simultaneous cell-mediated and humoral immune response acquisition. Immunized mice with positive delayed hypersensitivity (DTH) against C. albicans crude antigen showed significant protection against intravenous challenge with C. albicans. Furthermore, the transfer of immunoglobulins from rabbit anti-C. albicans serum to DTH-positive mice enhanced protection, while it did not protect control mice against a subsequent challenge with C. albicans. These results suggest that cell-mediated immunity plays a major role and humoral immunity a side role in the defense mechanism(s) of C. albicans infected mice.     

Life cycles of 17 riffle-dwelling mayfly species (Baetidae,Heptageniidae, and Ephemerellidae) in central Japan

We described the life cycles of 17 riffle-dwelling mayfly species in a central Japanese stream. Both species of Baetidae (Alainites yoshinensis Gose and Baetis thermicus Uéno) and two of nine species of Heptageniidae (Ecdyonurus scalaris Kluge and Epeorus latifolium Uéno/l-nigrus Matsumura) in this stream were multivoltine (more than one generation per year). Seven other heptageniid species (two unidentified species of Cinygmula, Epeorus aesculus Imanishi, E. curvatulus Matsumura, E. ikanonis Takahashi, E. napaeus Imanishi, and Rhithrogena japonica Uéno) and all six species of Ephemerellidae [Cincticostella elongatula (McLachlan), C. nigra (Uéno), Drunella basalis (Imanishi), D. ishiyamana Matsumura, D. sachalinensis (Matsumura), and D. trispina (Uéno)] were essentially univoltine.     

Sesamin and alpha-tocopherol synergistically suppress lipid-peroxide in rats fed a high docosahexaenoic acid diet

Docosahexaenoic acid (DHA) is an essential nutrient for human health, but has extremely high oxidative susceptibility. We examined the suppressing effect of sesamin, a sesame seed lignan, on lipidperoxides in rats fed a low alpha-tocopherol and high DHA containing diet. Groups of rats were fed four experimental diets: low alpha-tocopherol (10 mg/kg diet) control diet, low alpha-tocopherol + 0.2% sesamin diet, low alpha-tocopherol + 0.5% DHA diet and low alpha-tocopherol + 0.5% DHA + 0.2% sesamin diet. TBARS concentrations in plasma and liver were significantly increased by DHA, but were completely suppressed by sesamin. Alpha-tocopherol concentrations in plasma and liver decreased by addition of DHA, but with sesamin recovered to the control level. The addition of DHA into the diets caused remarkable increases of DHA concentrations in plasma and liver lipids. Sesamin caused a significant increase of DHA concentrations in the triacylglycerol of plasma.     

Physical size of the <Emphasis Type="Italic">S</Emphasis> locus region defined by genetic recombination and genome sequencing in <Emphasis Type="Italic">Ipomoea trifida</Emphasis>, Convolvulaceae

Sporophytic self-incompatibility (SSI) in the genus Ipomoea (Convolvulaceae) is controlled by a single polymorphic S locus. We have previously analyzed genomic sequences of an approximately 300 kb region spanning the S locus of the S ₁ haplotype and characterized the genomic structure around this locus. Here, we further define the physical size of the S locus region by mapping recombination breakpoints, based on sequence analysis of PCR fragments amplified from the genomic DNA of recombinants. From the recombination analysis, the S locus of the S ₁ haplotype was delimited to a 0.23 cM region of the linkage map, which corresponds to a maximum physical size of 212 kb. To analyze differences in genomic organization between S haplotypes, fosmid contigs spanning approximately 67 kb of the S ₁₀ haplotype were sequenced. Comparison with the S ₁ genomic sequence revealed that the S haplotype-specific divergent regions (SDRs) spanned 50.7 and 34.5 kb in the S ₁ and S ₁₀ haplotypes, respectively and that their flanking regions showed a high sequence similarity. In the sequenced region of the S ₁₀ haplotype, five of the 12 predicted open reading frames (ORFs) were found to be located in the divergent region and showed co-linear organization of genes between the two S haplotypes. Based on the size of the SDRs, the physical size of the S locus was estimated to fall within the range 34–50 kb in Ipomoea.     

Across-reach consistency in macroinvertebrate distributions among litter patch types in Japanese headwater streams

Different types of litter patches with contrasting macroinvertebrate assemblages have been observed within a stream reach. This study examined whether distributions of macroinvertebrates among three litter patch types (riffle, middle, edge) were consistent between reaches with different channel characteristics in headwater streams in central Japan. Mass of leaves per unit area was significantly higher in riffle and edge patches than in middle patches, which was consistent between reaches, while no consistent pattern was evident between reaches for mass of either woody material or small litter fragments. Distribution among the patch types was consistent between reaches for 11 out of 13 dominant macroinvertebrate taxa; density was highest in riffle patches for 5 taxa and in middle patches for 5 taxa. Although we previously related densities of some taxa to mass of woody material or small litter fragments, hydraulic characteristics (water depth, current velocity), which were consistent between reaches, may be more important determinants of macroinvertebrate distributions among the patch types, even within pools (i.e. middle and edge patches) where current is uniformly low. The results of this study indicate that a reach-scale macroinvertebrate community structure associated with litter is likely to vary according to litter patch type composition, which would be affected by channel characteristics of the reaches.     

Feeding by omnivores increases food available to consumers

Consumers are usually thought of as negatively affecting producers, but they can affect them positively by releasing nutrients (nutrient regeneration). The net effects of consumers on producers should depend on the balance between the effects of consumption and nutrient regeneration. In aquatic habitats, nutrient regeneration by consumers may increase microbial activity on leaf detritus as well as algal production, which in turn may stimulate further nutrient release and benefit herbivores or detritivores by increasing food quantity or quality. Omnivores can regenerate nutrients from animals, algae and detritus, creating diverse nutrient pathways. Many tadpoles are omnivores, and their nutrient regeneration may be important in aquatic food webs. To reveal the nutrient pathways created by tadpoles and examine whether omnivorous tadpoles can have positive effects on producers and consumers, we experimentally examined the effects of nutrient regeneration by three densities of tadpoles on primary producers, leaf litter, and other consumers in tank mesocosms. Tadpole exclosures were placed inside each mesocosm, allowing us to separate direct consumption effects from indirect nutrient regeneration effects. Nutrient regeneration caused by the herbivorous and carnivorous feeding activities of tadpoles positively affected rates of production of benthic algae, phytoplankton, and herbivorous benthic chironomid larvae, and rates of mineralization of leaf litter. The increased production of benthic algae and chironomid larvae was consumed by the tadpoles themselves, leaving no net change in the standing biomass of these resources. Our experiment thus demonstrated that omnivores created complicated nutrient pathways and accelerated rates of primary production and growth rates of other consumers, leading to increased rates of food availability to the omnivores themselves. Interactions of this nature may be common in many systems and could strongly moderate the effects of consumers on their resources and each other.