Mitochondrial DNA Variation Follows a Geographic Pattern in Japanese Beech Species

The amount and distribution of mitochondrial (mt) DNA restriction fragment length polymorphism was determined among individual tree samples of two Japanese beech species, Fagus crenata and F.japonica. Individual plants were collected from 16 F. crenata populations throughout the range of the species, and from three F. japonica populations. We detected enough variation to characterize eleven and three chondriome types in F. crenata and F.japonica, respectively. The grouping of beech chondriome types based upon the cladistic analysis of mtDNA polymorphism allowed us to recognize the apparent geographical patterns of mtDIMA diversity: the resulting three main groups occupied distinct geographic areas. This geographic differentiation is likely to reflect the history of the Japanese beech forests after the last glacial period of the Pleistocene. In addition, the mtDNA polymorphism encountered within F. crenata encompassed all the variation observed in F.japonica. Our result suggests the need for re-evaluation of their phylogenetic relationships.     

Induction of ornithine decarboxylase by sialagogues in a human parotid gland adenocarcinoma cell line.

Ornithine decarboxylase in a human parotid gland adenocarcinoma cell line was induced by both cholinergic (carbachol) and beta-adrenergic (isoproterenol) sialagogues. The enzyme protein level, measured with anti-peptide antiserum, as well as the enzyme activity, was found to be high in unstimulated cells and to increase approximately 2-fold on stimulation, while the mRNA level increased 3-4 fold, as revealed by Northern hybridization. The rise in activity was completely blocked by the simultaneous addition of antagonists or actinomycin D. These results suggest that receptor-mediated stimulation of ornithine decarboxylase activity by sialagogues involves alterations in the level of mRNA and that the proliferative responses of human parotid cells to these sialagogues resemble those of the murine parotid gland.     

Expanding Imaging Capabilities for Microfluidics: Applicability of Darkfield Internal Reflection Illumination (DIRI) to Observations in Microfluidics

Microfluidics is used increasingly for engineering and biomedical applications due to recent advances in microfabrication technologies. Visualization of bubbles, tracer particles, and cells in a microfluidic device is important for designing a device and analyzing results. However, with conventional methods, it is difficult to observe the channel geometry and such particles simultaneously. To overcome this limitation, we developed a Darkfield Internal Reflection Illumination (DIRI) system that improved the drawbacks of a conventional darkfield illuminator. This study was performed to investigate its utility in the field of microfluidics. The results showed that the developed system could clearly visualize both microbubbles and the channel wall by utilizing brightfield and DIRI illumination simultaneously. The methodology is useful not only for static phenomena, such as clogging, but also for dynamic phenomena, such as the detection of bubbles flowing in a channel. The system was also applied to simultaneous fluorescence and DIRI imaging. Fluorescent tracer beads and channel walls were observed clearly, which may be an advantage for future microparticle image velocimetry (μPIV) analysis, especially near a wall. Two types of cell stained with different colors, and the channel wall, can be recognized using the combined confocal and DIRI system. Whole-slide imaging was also conducted successfully using this system. The tiling function significantly expands the observing area of microfluidics. The developed system will be useful for a wide variety of engineering and biomedical applications for the growing field of microfluidics.     

Mitigation of copper toxicity by DNA oligomers in green paramecia

Impact of transition metals which catalyze the generation of reactive oxygen species (ROS), on activation of cell death signaling in plant cells have been documented to date. Similarly in green paramecia (Paramecium bursaria), an aquatic protozoan species harboring symbiotic green algae in the cytoplasm, toxicities of various metallic ions have been documented. We have recently examined the effects of double-stranded GC-rich DNA fragments with copper-binding nature and ROS removal catalytic activity as novel plant cell-protecting agents, using the suspension-cultured tobacco cells. Here, we show that above DNA oligomers protect the cells of green paramecia from copper-induced cell death, suggesting that the phenomenon firstly observed in tobacco cells is not limited only within higher plants but it could be universally observable in wider range of organisms.     

Relationships between Micro-Vascular and Iodine-Staining Patterns in the Vicinity of the Tumor Front of Superficial Esophageal Squamous Carcinoma

Objective

The aim of the present study was to clarify differences between micro-vascular and iodine-staining patterns in the vicinity of the tumor fronts of superficial esophageal squamous cell carcinomas (ESCCs).

Methods

Ten consecutive patients with ESCCs who were treated by endoscopic submucosal dissection (ESD) were enrolled. At the edge of the iodine-unstained area, we observed 183 sites in total using image-enhanced magnifying endoscopy. We classified the micro-vascular and iodine-staining patterns into three types: Type A, in which the line of vascular change matched the border of the iodine-unstained area; Type B, in which the border of the iodine-unstained area extended beyond the line of vascular change; Type C, in which the line of vascular change extended beyond the border of the iodine-unstained area. Then, by examining histopathological sections, we compared the diameter of intra-papillary capillary loops (IPCLs) in cancerous areas and normal squamous epithelium.

Results

We investigated 160 sites that the adequate quality of pictures were obtained. There was no case in which the line of vascular change completely matched the whole circumference of the border of an iodine-unstained area. Among the 160 sites, type A was recognized at 76 sites (47.5%), type B at 79 sites (49.4%), and type C at 5 sites (3.1%). Histological examination showed that the mean diameter of the IPCLs in normal squamous epithelium was 16.2±3.7μm, whereas that of IPCLs in cancerous lesions was 21.0±4.4μm.

Conclusions

The development of iodine-unstained areas tends to precede any changes in the vascularity of the esophageal surface epithelium.     

Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli

Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (K _m = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (K _m = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.     

NMR analyses on the interactions of the yeast Tim50 C-terminal region with the presequence and Tim50 core domain

The mitochondrial targeting signal in the presequence of mitochondrial precursor proteins is recognized by Tom20 and subsequently by Tim50 in mitochondria. Yeast Tim50 contains two presequence binding sites in the conserved core domain and in the fungi-specific C-terminal presequence binding domain (PBD). We report the NMR analyses on interactions of a shorter variant of PBD (sPBD), a shorter variant of PBD, with presequences. The presequence is recognized by sPBD in a similar manner to Tom20. sPBD can also bind to the core domain of Tim50 through the presequence binding region, which could promote transfer of the presequence from sPBD to the core domain in Tim50.     

Structure–activity relationship of a novel pentapeptide with cancer cell growth‐inhibitory activity

We previously reported that yamamarin, a pentapeptide with an amidated C‐terminus (DILRG‐NH₂) isolated from larvae of the silkmoth, and its palmitoylated analog (C16‐DILRG‐NH₂) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure–activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C‐terminal part (‐RG‐NH₂) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG‐NH₂ and C16‐DILRG‐NH₂ revealed that the N‐terminal palmitoyl group of C16‐DILRG‐NH₂ did not affect the conformation of the C‐terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A green Paramecium strain with abnormal growth of symbiotic algae

Some hundred cells of Chlorella-like green algae are naturally enclosed within the cytoplasm of a single cell of green paramecia (Paramecium bursaria). Therefore, P. bursaria serves as an experimental model for studying the nature of endo-symbiosis made up through chemical communication between the symbiotic partners. For studying the mechanism of symbiotic regulations, the materials showing successful symbiosis are widely used. Apart from such successful model materials, some models for symbiotic distortion would be of great interest in order to understand the nature of successful symbiosis. Here, we describe a case of unsuccessful symbiosis causing unregulated growth of algae inside the hosting ciliates. Recently, we have screened some cell lines, from the mass of P. bursaria cells survived after paraquat treatment. The resultant cell lines (designated as KMZ series) show novel and unusual morphological features with heavily darker green colour distinguishable from the original pale green-coloured paramecia. In this type of isolates, endo-symbiotic algae are restricted within one or two dense spherical structures located at the center of the host cells' cytoplasm. Interestingly, this isolate maintains the host cells' circadian mating response which is known as an alga-dependent behaviour in the host cells. In contrast, we discuss that KMZ lacks the host-dependent regulation of algal growth, thus the algal complex often over-grows obviously exceeding the original size of the normal hosting ciliates. Additionally, possible use of this isolate as a novel model for symbiotic cell-to-cell communication is discussed.     

Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.