HuC:Kaede, a useful tool to label neural morphologies in networks in vivo

A simple and efficient procedure for labeling neurons is a prerequisite for investigating the development of neural networks in zebrafish. To label neurons we used Kaede, a fluorescent protein with a photoconversion property allowing conversion from green to red fluorescence following irradiation with UV or violet light. We established a zebrafish stable transgenic line, Tg(HuC:Kaede), expressing Kaede in neurons under the control of the HuC promoter. This transgenic line was used to label a small number of neurons in the trigeminal ganglion. Also, using embryos injected with the transgene, we labeled peripheral axon arbors of a Rohon-Beard neuron at 4 days postfertilization and observed the dendrite development of a tectal neuron for 3 days. These data indicate that Kaede is a useful tool to selectively label neural networks in zebrafish.     

An electrophoretic karyotype for Schizosaccharomyces pombe by pulsed field gel electrophoresis.

The three chromosomal DNAs of S. pombe have been fractionated by pulsed field gel electrophoresis. The resulting molecular karyotype will greatly speed gene mapping in this organism, and it indicates that the separation range of the technique extends to DNA molecules as large as 9,000,000 base pairs.     

Role of spinal voltage-dependent calcium channel alpha 2 delta-1 subunit in the expression of a neuropathic pain-like state in mice

The present study was undertaken to investigate the role of spinal voltage-dependent calcium channel alpha(2)delta-1 subunit in the expression of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. In cultured spinal neurons, gabapentin (GBP), which displays the inhibitory effect of alpha(2)delta-1 subunit, suppressed the extracellular Ca(2+) influx induced by KCl, whereas it failed to inhibit the intracellular Ca(2+) release induced by inositol-1,4,5-triphosphate. Seven days after sciatic nerve ligation, the protein level of alpha(2)delta-1 subunit in the ipsilateral spinal cord was clearly increased compared to that observed in sham-operated mice. In addition, the mRNA level of alpha(2)delta-1 subunit was significantly increased in the dorsal root ganglion, but not in the spinal cord, of nerve-ligated mice. Under these conditions, a marked decrease in the latency of paw-withdrawal against a thermal stimulation and tactile stimulation, induced by sciatic nerve ligation was abolished by repeated intrathecal (i.t.) treatment with GBP. Additionally, the persistent reduction in the nociceptive threshold by i.t. treatment with GBP at the early stage of the neuropathic pain-like state was maintained for 7 days even after GBP withdrawal. It is of interest to note that a single i.t. post-injection of GBP showed a marked and transient inhibitory effect on the developed neuropathic pain-like state, whereas repeated i.t. post-treatment with GBP produced a persistent inhibitory effect during the treatment. In conclusion, we propose here that the neuropathic pain-like state with sciatic nerve ligation is associated with the increased level of the alpha(2)delta-1 subunit of Ca(2+) channels at the sensory nerve terminal in the spinal dorsal horn of mice. Furthermore, the present data provide evidence that the neuropathic pain may be effectively controlled by repeated treatment with GBP at the early stage.     

Organ-Level Analysis of Idioblast Patterning in Egeria densa Planch. Leaves

Leaf tissues of plants usually contain several types of idioblasts, defined as specialized cells whose shape and contents differ from the surrounding homogeneous cells. The spatial patterning of idioblasts, particularly of trichomes and guard cells, across the leaf epidermis has received considerable attention as it offers a useful biological model for studying the intercellular regulation of cell fate and patterning. Excretory idioblasts in the leaves of the aquatic monocotyledonous plant Egeria densa produced light blue autofluorescence when irradiated with ultraviolet light. The use of epifluorescence microscopy to detect this autofluorescence provided a simple and convenient method for detecting excretory idioblasts and allowed tracking of those cells across the leaf surfaces, enabling quantitative measurement of the clustering and spacing patterns of idioblasts at the whole leaf level. Occurrence of idioblasts was coordinated along the proximal–distal, medial–lateral, and adaxial–abaxial axes, producing a recognizable consensus spatial pattern of idioblast formation among fully expanded leaves. Idioblast clusters, which comprised up to nine cells aligned along the proximal–distal axis, showed no positional bias or regularity in idioblast-forming areas when compared with singlet idioblasts. Up to 75% of idioblasts existed as clusters on every leaf side examined. The idioblast-forming areas varied between leaves, implying phenotypic plasticity. Furthermore, in young expanding leaves, autofluorescence was occasionally detected in a single giant vesicle or else in one or more small vesicles, which eventually grew to occupy a large portion of the idioblast volume as a central vacuole. Differentiation of vacuoles by accumulating the fluorescence substance might be an integral part of idioblast differentiation. Red autofluorescence from chloroplasts was not detected in idioblasts of young expanding leaves, suggesting idioblast differentiation involves an arrest in chloroplast development at a very early stage, rather than transdifferentiation of chloroplast-containing epidermal cells.     

Congenital neuroglial heterotopia in a neonatal harbor seal (Phoca vitulina richardsi ) with evidence of recent exposure to polycyclic aromatic hydrocarbons

A male neonatal Pacific harbor seal (Phoca vitulina richardsi) stranded off the coast of California, USA, was presented for rehabilitation with numerous partially haired, soft tissue masses around the mouth and in the oropharynx. Because of the extent of the lesions, the seal was humanely euthanized. Histologically, the masses consisted of subepithelial connective tissue and subcutis expanded by a proliferation of streams and bundles of spindle to stellate cells. Morphology of these cells suggested a neural origin, which was confirmed by positive immunohistochemistry for two neural markers, S-100 protein and glial fibrillary acidic protein, so the masses were diagnosed as neuroglial heterotopia. Heterotopic neuroglial tissue is a rare lesion comprised of benign mature neural tissue in an ectopic location with no connection to the central nervous system. Results of polycyclic aromatic hydrocarbon (PAH) metabolite analysis of bile indicated recent exposure to a petroleum source. Although fetal exposure to PAHs in utero can cause neurotoxicity and affect normal embryonic development, it is unknown whether gestational exposure occurred in this case.     

Glucosylceramide Contained in Koji Mold-Cultured Cereal Confers Membrane and Flavor Modification and Stress Tolerance to Saccharomyces cerevisiae during Coculture Fermentation

In nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeast Saccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured with Aspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer from A. kawachii modifies the physiology of the yeast S. cerevisiae and demonstrate a new mechanism for cooperation between microbes in food fermentation.     

Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase II

Fission Yeast DNA topoisomerase II (165 kD) consists of an enzymatically active 125-kD core, approximately 10-kD NH2-terminal and 30-kD COOH-terminal domains. The question addressed in the present study is what is the role of the topo II termini. Although deletion of either the NH2 or the COOH terminus is viable, deletion of both termini is lethal; the termini share an essential role for viability. We show here that topo II phosphorylation sites are localized in the terminal domains, but dephosphorylated topo II is still active. The topo II terminal sequences are required for nuclear localization; topo II double terminal deletion mutants are deficient for nuclear targeting, whereas wild-type and single deletion mutant topo IIs are transported into the nucleus with different efficiencies. Functional subdomains in the NH2 terminus are further dissected; we identified a 15 amino acid nuclear localization sequence (NLS) which is essential for viability and nuclear localization when the COOH terminus is deleted. This NLS could be substituted with SV-40 large T-antigen NLS. Two other functional subdomains were found; a non-essential acidic stretch which is phosphorylated and apparently enhances the nuclear localization and an essential hydrophilic stretch of unknown function. Motifs similar to these three NH2-terminal subdomains are also found in the COOH terminus. Our results support the possibility that phosphorylation of topo II does not play an essential role in fission yeast.     

Phylogenetic relationships within Echinococcus and Taenia tapeworms (Cestoda: Taeniidae): an inference from nuclear protein-coding genes

The family Taeniidae of tapeworms is composed of two genera, Echinococcus and Taenia, which obligately parasitize mammals including humans. Inferring phylogeny via molecular markers is the only way to trace back their evolutionary histories. However, molecular dating approaches are lacking so far. Here we established new markers from nuclear protein-coding genes for RNA polymerase II second largest subunit (rpb2), phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold). Bayesian inference and maximum likelihood analyses of the concatenated gene sequences allowed us to reconstruct phylogenetic trees for taeniid parasites. The tree topologies clearly demonstrated that Taenia is paraphyletic and that the clade of Echinococcus oligarthrus and Echinococcusvogeli is sister to all other members of Echinococcus. Both species are endemic in Central and South America, and their definitive hosts originated from carnivores that immigrated from North America after the formation of the Panamanian land bridge about 3 million years ago (Ma). A time-calibrated phylogeny was estimated by a Bayesian relaxed-clock method based on the assumption that the most recent common ancestor of E. oligarthrus and E. vogeli existed during the late Pliocene (3.0 Ma). The results suggest that a clade of Taenia including human-pathogenic species diversified primarily in the late Miocene (11.2 Ma), whereas Echinococcus started to diversify later, in the end of the Miocene (5.8 Ma). Close genetic relationships among the members of Echinococcus imply that the genus is a young group in which speciation and global radiation occurred rapidly.     

The secretory response through electric stimulation of differentiated PC12 rat pheochromocytoma cells transfected with neuropeptide Y fused with enhanced green fluorescent protein

Exocytosis in pheochromocytoma cells was induced by electric stimulation. To chase the movement of vesicles by electric stimulation, dense-core secretory vesicles were visualized by expression of the fusion protein between neuropeptide Y and enhanced green fluorescent protein (EGFP) in these differentiated PC12 rat pheochromocytoma cells. When the cells were stimulated with constant voltage potential at –300 mV, the movement of dense-core secretory vesicles could be regulated.