Methylcytosine-selective fluorescence quenching by osmium complexation

We report on the control of the emission from a fluorophore fixed on DNA using the methylcytosine-selective addition of an osmium-bipyridine complex. We have synthesized DNA modified by a microenvironment-sensitive fluorophore, 2-dimethylamino-6-acyl-naphthalene. The emission from the fluorophore tethered to a probe DNA was effectively quenched by a methylcytosine glycol-osmium-bipyridine triad, which was located in the immediate neighborhood of the fluorophore. The discrimination of the cytosine methylation status at a methylation hot spot in the p53 gene was also executed using a well-designed fluorescent DNA probe.     

A familial case of visceral toxocariasis due to consumption of raw bovine liver

We present 3 adult cases of visceral toxocariasis from the same family, who each consumed thin slices of raw bovine liver weekly, and developed eosinophilia and multiple small lesions in their livers and lungs. Serological examinations using the larval excretory–secretory product of Toxocara canis strongly indicated infection with Toxocara species larvae. The patients responded well to treatment with albendazole. Ingestion of raw liver from paratenic animals is considered to be a common transmission route of human toxocariasis, especially in adults.     

Ancient divergence of animal protein tyrosine kinase genes demonstrated by a gene family tree including choanoflagellate genes

Animal-specific gene families involved in cell-cell communication and developmental control comprise many subfamilies with distinct domain structures and functions. They diverged by subfamily-generating duplications and domain shufflings before the parazoan-eumetazoan split. Here, we have cloned 40 PTK cDNAs from choanoflagellates, Monosiga ovata, Stephanoeca diplocostata and Codosiga gracilis, the closest relatives to animals. A phylogeny-based analysis of PTKs revealed that 40 out of 47 subfamilies analyzed have unique domain structures and are possibly generated independently in animal and choanoflagellate lineages by domain shufflings. Seven cytoplasmic subfamilies showed divergence before the animal-choanoflagellate split originated by both duplications and shufflings.     

The susceptibility of pregnant mice to encephalomyocarditis (EMC) virus infection on different days of gestation.

Pregnant mice of the BALB/c CrSlc strain were experimentally infected with the D variant of encephalomyocarditis virus (EMC-D, 5 x 10(2) PFU/head) on three different gestational days (GD). Mice were intraperitoneally inoculated with EMC-D on 11, 13 and 15 GD and sacrificed 3 days post inoculation. There was no significant difference in the fetal mortality among all inoculation groups. Placenta showed higher virus titer than fetus and dam's serum in all inoculation groups, and the virus titer of the fetus was lowest in the 15GD group. Histopathological changes and signals of viral RNAs detected by in situ hybridization were observed almost restricted to the spongiotrophoblast layer of the placenta in all inoculation groups, and the signals were strongest in the 11GD group. In the fetus of the11GD group, signals of viral RNAs were also seen in myocardium and hepatocytes. Ultrastructurally, intracytoplasmic aggregations of virus-like particles in crystalline array were observed in trophoblast cells and giant cells in the spongiotrophoblast layer in all inoculation groups.     

Accurate Determination of S-Phase Fraction in Proliferative Cells by Dual Fluorescence and Peroxidase Immunohistochemistry with 5-Bromo-2��-Deoxyuridine (BrdU) and Ki67 Antibodies

To ensure the maintenance of tissues in mammals, cell loss must be balanced with cell production, the proliferative activity being different from tissue to tissue. In this article, the authors propose a new method for the quantification of the proliferative activity, defined as the S-phase fraction of actively cycling cells, by dual labeling with fluorescence and peroxidase immunohistochemistry using BrdU (marker of S-phase) and Ki67 antibodies (marker of G₁-, S-, G₂-, and M-phases) after a one-step antigen retrieval. In the generative cell zones of fundic and pyloric glandular stomachs, where the majority of cells were cycling, the authors measured a proliferative activity of 31%. In the epithelium of the forestomach and the skin, where cycling cells are intermingled with G₀ and differentiated cells, proliferative activities were 21% and 13%, respectively. In the adrenal cortex, in which cycling cells were sparsely distributed, the proliferative activity reached 32%. During the regenerative process in the skin after a lesion, the proliferative activity increased in proximity to the wound. The present one-step dual-labeling method has revealed that the proliferative activity is different between tissues and depends on the physiological or pathological state.     

Multiple Skp1-related proteins in Caenorhabditis elegans: diverse patterns of interaction with Cullins and F-box proteins

BACKGROUND: The ubiquitin-proteasome pathway of proteolysis controls the abundance of specific regulatory proteins. The SCF complex is a type of ubiquitin-protein ligase (E3) that contributes to this pathway in many biological systems. In yeast and mammals, the SCF complex consists of common components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Whereas only one functional Skp1 gene is present in the human genome, the genome of Caenorhabditis elegans has now been shown to contain at least 21 Skp1-related (skr) genes. The biochemical properties, expression, and function of the C. elegans SKR proteins were examined. RESULTS: Of the 17 SKR proteins examined, eight (SKR-1, -2, -3, -4, -7, -8, -9, and -10) were shown to interact with C. elegans CUL1 by yeast two-hybrid analysis or a coimmunoprecipitation assay in mammalian cells. Furthermore, SKR proteins exhibited diverse binding specificities for C. elegans F-box proteins. The tissue specificity of expression of the CUL1-interacting SKR proteins was also varied. Suppression of skr-1 or skr-2 genes by double-stranded RNA interference resulted in embryonic death, whereas that of skr-7, -8, -9, or -10 was associated with slow growth and morphological abnormalities. CONCLUSIONS: The multiple C. elegans SKR proteins exhibit marked differences in their association with Cullins and F-box proteins, in tissue specificity of expression, and in phenotypes associated with functional suppression by RNAi. At least eight of the SKR proteins may, like F-box proteins, act as variable components of the SCF complex in C. elegans.     

Ploidy distribution in the explant tissue and the calluses induced during the initial stage of internode segment culture of Asparagus officinalis L.

Summary To pursue the causal factors of ploidy variation in tissue culture of asparagus (Asparagus officinalis L.), ploidy status of stem explants and the calluses induced from these explants were examined using flow cytometry (FCM). The frequency of higher ploidy (>8C) cells in the stem explants increased with increasing distance from the shoot apex. Calluses derived from sections far from the apex also showed higher ploidy distributions, and also higher ploidy (16C) in general than was observed in the explant tissue. Detailed studies using FCM and charge-coupled device (CCD)-equipped fluorescence microscopy showed that 16C cells appeared in the explant tissues within 1 wk of culture, possibly induced by the plant growth regulators (PGRs), especially 1-naphthaleneacetic acid (NAA), in the culture medium.     

SELECTION FOR PRIME-NUMBER INTERVALS IN A NUMERICAL MODEL OF PERIODICAL CICADA EVOLUTION

Periodical cicadas are known for unusually long and prime-numbered life cycles (13 and 17 years) for insects. To explain the evolution of prime-numbered reproductive intervals (life cycles), the hybridization hypothesis claims that prime numbers greatly reduce the chance of hybridization with other life cycles. We investigate the hybridization hypothesis using a simulation model. This model is a deterministic, discrete population model with three parameters: larval survival per year, clutch size, and emergence success. Reproductive intervals from 10 years to 20 years compete for survival in the simulations. The model makes three key assumptions: a Mendelian genetic system, random mating among broods of different life-cycle lengths, and integer population sizes. Longer life cycles have larger clutch sizes but suffer higher total mortality than shorter life cycles. Our results show that (1) nonprime-numbered reproductive intervals disappear rapidly in comparison to the selection among the various prime-numbered life cycles, (2) the selection of prime-numbered intervals happens only when populations are at the verge of extinction, and (3) the 13- and 17-year prime phenotypes evolve under certain conditions of the model and may coexist. The hybridization hypothesis is discussed in light of other hypotheses for the evolution of periodical cicada life cycles.     

Nitrogen-assimilating enzymes in land plants and algae: phylogenic and physiological perspectives

An important biochemical feature of autotrophs, land plants and algae, is their incorporation of inorganic nitrogen, nitrate and ammonium, into the carbon skeleton. Nitrate and ammonium are converted into glutamine and glutamate to produce organic nitrogen compounds, for example proteins and nucleic acids. Ammonium is not only a preferred nitrogen source but also a key metabolite, situated at the junction between carbon metabolism and nitrogen assimilation, because nitrogen compounds can choose an alternative pathway according to the stages of their growth and environmental conditions. The enzymes involved in the reactions are nitrate reductase (EC 1.6.6.1-2), nitrite reductase (EC 1.7.7.1), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.13-14, 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.2-4), aspartate aminotransferase (EC 2.6.1.1), asparagine synthase (EC 6.3.5.4), and phosphoenolpyruvate carboxylase (EC 4.1.1.31). Many of these enzymes exist in multiple forms in different subcellular compartments within different organs and tissues, and play sometimes overlapping and sometimes distinctive roles. Here, we summarize the biochemical characteristics and the physiological roles of these enzymes. We also analyse the molecular evolution of glutamine synthetase, glutamate synthase and glutamate dehydrogenase, and discuss the evolutionary relationships of these three enzymes.