Nano-mechanical properties of living cells expressing constitutively active RhoA effectors

Filamentous actin and myosin-II are major determinants of cell mechanics and are tightly regulated by a small guanosine triphosphatase, RhoA, and its downstream effectors. We examined the effects of constitutively active mutants of RhoA effectors, which have not been reported before, on cortical stiffness of living cells by using scanning probe microscopy, fluorescence microscopy, and truncated mutants of RhoA effectors labeled with a fluorescent protein. Our data indicated that expression of a constitutively active mutant of Dia1, a formin-family actin polymerizer, enhanced cortical stiffness and increased actin filament quantity in cells. Furthermore, expression of a constitutively active mutant of Rho-associated coiled-coil kinase, a myosin-II activator, softened the cell cortex but increased myosin-II activity. Our findings provide new insights into anomalous mechanics of cells, which is a topic of current interest in a variety of biological research fields.     

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces to biological hydrogels for rapid and efficient cell micropatterning

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces onto biological hydrogel sheets is investigated herein, as a new cell stamping method for both cell microarray and tissue engineering. By overlaying a hydrogel sheet on the cells immobilized on the poly(ethylene glycol)-oleyl surface and successively peeling it off, the immobilized cells were transferred onto a hydrogel sheet because the adhesive interaction between the cells and the hydrogel was stronger than that between the cells and the poly(ethylene glycol)-oleyl surface. Four types of human cell could be efficiently transferred onto a rigid collagen sheet. The transfer printing ratios, for all cells, were above 80% and achieved within 90 min. A cell microarray was successfully prepared on a collagen gel sheet using the present stamping method. We have also demonstrated that the transferred pattern of endothelial cells is transformed to the patterned tube-like structure on the reconstituted basement membrane matrix. Finally, the patterns of two types of endothelial cell are shown to be easily prepared on the matrix, and the desired tube-like structures, including the orderly pattern of the two different cells, were formed spontaneously. Thus, the present poly(ethylene glycol)-oleyl coated substrates are useful for rapid and efficient cell stamping, in the preparation of multi-cellular pattern on extracellular matrices.     

The role of the C-terminal region of rat brain natriuretic peptide in receptor selectivity.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have different C-terminal tail structures compared with the rather conservative ring structures which consist of 17 amino acid residues. To examine the different effects of the tail structures of ANP and BNP on their interaction with receptors, we synthesized several peptide analogs and measured their biological actions in three different assay systems. Deletion of the C-terminal tail from rat BNP did not effect the vasorelaxation activity against rat aorta, but it promoted cGMP production in cultured rat aortic smooth muscle cells (RASMC). Deletion of the C-terminal tail from rat ANP diminished both vasorelaxant and cGMP producing activities. In a binding competition assay with RASMC and [125I]rat ANP-(1-28), the competition activities of both ANP and BNP were greatly reduced by C-terminal deletion. In addition, we obtained agonists with novel receptor selectivity.     

Surveillance of fish species composition using environmental DNA

Prompt and accurate methods for assessing the species composition of given areas are indispensable in addressing the rapid loss of biodiversity. Here, we propose a method for the surveillance of fish species composition in freshwater using environmental DNA as species markers. First, the applicability of the method was demonstrated through aquarium experiments. DNA was extracted from 120?ml aquarium water, and the degenerated primers targeting the fish mitochondrial cytochrome b gene were used for amplification. PCR-amplified fragments were analysed by random cloning, and all species reared in the aquarium were detected. Next, this method was applied to natural freshwater environments. Water samples were collected from three sites in the Yura River, Japan; DNA was concentrated from 2?l of environmental water, and then amplified and cloned. Up to four species of fish were detected by sequencing 47 randomly selected clones from a single water sample. Overall, the results were consistent with previous knowledge of fish habitat utilisation. Using this method, the surveillance of fish species composition can be conducted less laboriously than with traditional methods.     

Relationship between the obstacle height cognition and step movement in the elderly

Background

This study examines the effect of obstacle height cognition (OHC) on single-leg forward step (SFS) and Obstacle-SFS.

Methods

In the SFS test, participants stepped 25 cm forward with one leg and returned it to its original position five times as quickly as possible. The Obstacle-SFS added an obstacle to the above condition in the SFS test. The participants were divided into two groups: tripping group, which tripped over an obstacle in the Obstacle-SFS test; and non-tripping group, which did not trip. Parameters were step time (T), the time it took to step forward (F), and the time it took to return to the original position (R). The OHC was determined by the difference between the elevated leg’s height and the obstacle height (10 cm), which was set at 60 cm in front of the participant.

Results

OHC showed a significant and moderate relationship with all parameters of Obstacle-SFS (OSFS-T, OSFS-F and OSFS-R). The tripping group had significantly larger values in the OHC, OSFS-T and OSFS-F than the non-tripping group.

Conclusions

In conclusion, the differences in obstacle height cognition ability may affect Obstacle-SFS movement.     

Screening and identification of disaccharides with insulin mimetic activity against L6 cells

Insulin mimetics are considered as prospective anti-diabetic agents, and the disaccharide, neohesperidose, has been found to show insulin mimetic activity against L6 cells. We screened several other disaccharides for their insulin mimetic activity and identified three new insulin mimetic disaccharides.     

Survey of Babesia microti infection in field rodents in Japan: records of the Kobe-type in new foci and findings of a new type related to the Otsu-type

Of 247 rodents comprising 5 genera and 7 species collected at 17 sites throughout Japan from 2003 to 2005, Babesia microti was detected microscopically and by polymerase chain reaction (PCR) in 36 rodents comprising 2 genera and 3 species from 12 sites. Based on the analysis of small subunit ribosomal RNA gene (SSUrDNA) sequences, the Kobe-type, the etiological type of the first Japanese case of human infection was found in Apodemus speciosus and Apodemus argenteus in Aomori, the northernmost prefecture of the Japanese mainland, while the U.S.-type was found on Hokkaido Island and the Otsu-type was widely distributed. In addition, a new Otsu-related type was detected exclusively in Eothenomys andersoni in Nagano, a prefecture in central Japan. The sequences of internal transcribed spacer 1 to 2 (ITS1/2) of the present Kobe- and Otsu-types were almost identical to those of the same types previously identified. The ITS1/2 sequence of the U.S.-type identified in Hokkaido in this survey was somewhat different from that of the U.S.-type strain originating from the U.S.A., with approximately 95% identity. This value was similar to the 94% identity found between the ITS1/2 sequences of the Otsu-type and the new Otsu-related type. The new Otsu-related type of B. microti was isolated as the Nagano strain, which was serologically differentiated from the other type strains of B. microti. The divergence and distribution of genotypes are important factors in investigating the epidemiology of human B. microti infection in Japan.     

Tick Surveillance for Relapsing Fever Spirochete Borrelia miyamotoi in Hokkaido,Japan

During 2012–2013, a total of 4325 host-seeking adult ticks belonging to the genus Ixodes were collected from various localities of Hokkaido, the northernmost island of Japan. Tick lysates were subjected to real-time PCR assay to detect borrelial infection. The assay was designed for specific detection of the Relapsing fever spirochete Borrelia miyamotoi and for unspecific detection of Lyme disease-related spirochetes. Overall prevalence of B. miyamotoi was 2% (71/3532) in Ixodes persulcatus, 4.3% (5/117) in Ixodes pavlovskyi and 0.1% (1/676) in Ixodes ovatus. The prevalence in I. persulcatus and I. pavlovskyi ticks were significantly higher than in I. ovatus. Co-infections with Lyme disease-related spirochetes were found in all of the tick species. During this investigation, we obtained 6 isolates of B. miyamotoi from I. persulcatus and I. pavlovskyi by culture in BSK-M medium. Phylogenetic trees of B. miyamotoi inferred from each of 3 housekeeping genes (glpQ, 16S rDNA, and flaB) demonstrated that the Hokkaido isolates were clustered with Russian B. miyamotoi, but were distinguishable from North American and European B. miyamotoi. A multilocus sequence analysis using 8 genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) suggested that all Japanese B. miyamotoi isolates, including past isolates, were genetically clonal, although these were isolated from different tick and vertebrate sources. From these results, B. miyamotoi-infected ticks are widely distributed throughout Hokkaido. Female I. persulcatus are responsible for most human tick-bites, thereby I. persulcatus is likely the most important vector of indigenous relapsing fever from tick bites in Hokkaido.     

Hydroxylamine enhances glucose uptake in C2C12 skeletal muscle cells through the activation of insulin receptor substrate 1

Diabetes mellitus is a global disease, and the number of patients with it is increasing. Of various agents for treatment, those that directly act on muscle are currently attracting attention because muscle is one of the main tissues in the human body, and its metabolism is decreased in type II diabetes. In this study, we found that hydroxylamine (HA) enhances glucose uptake in C2C12 myotubes. Analysis of HA’s mechanism revealed the involvement of IRS1, PI3 K and Akt that is related to the insulin signaling pathway. Further investigation about the activation mechanism of insulin receptor or IRS1 by HA may provide a way to develop a novel anti-diabetic agent alternating to insulin.     

Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis

Implementation of the on-chip immunoassay for α-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10 min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma, was achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFP-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis. Laser-induced-fluorescence (LIF) detection was used to quantitate the labeled immunocomplexes. The limit of detection (LOD) was 0.1 ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922 ng/ml total AFP in spiked serum samples. Assay precision of less than 3% CV was obtained for AFP-L3% measurements from 8.5 to 81%. Furthermore, good correlation of test results for 68 patient serum samples with a commercially available reference method (LiBASys assay for AFP-L3%) was obtained, with r² = 0.981 and slope = 1.03.