Functional glass surface displaying a glutamyl donor substrate for transglutaminase-mediated protein immobilization

A chemically modified glass surface displaying a glutamyl donor substrate peptide (Z-QG) was developed for microbial transglutaminase (MTG)-mediated immobilization of recombinant proteins tagged with an MTG-reactive lysine-containing substrate peptide (K-tag). To evaluate the surface modification conditions affecting the enzymatic protein immobilization, we employed an amino-modified 96-well glass plate as a base and prepared three types of glass surfaces displaying Z-QG. Validation of the Z-QG modified glass surfaces with recombinant enhanced green fluorescent proteins revealed that the insertion of a di(ethylene glycol) linker between the terminal Z-QG moiety and the base not only enhances enzymatic protein immobilization efficiency but also decreases nonselective protein adsorption. A bacterial alkaline phosphatase fused with a K-tag at the N terminus was also successfully immobilized to the designed glass surface, suggesting that the chemically modified glass surface displaying a spatially controlled glutamyl donor substrate is a potential platform for MTG-mediated fabrication of protein-based solid biomaterials.     

Preparation of connexin43‐integrated giant Liposomes by a baculovirus expression–liposome fusion method

Connexin‐43 (Cx43) containing giant liposomes (GL) were prepared by a baculovirus expression–liposome fusion method. Recombinant budded viruses expressing Cx43 were prepared and then fused with GLs containing DOPG/DOPC at pH 4.5. Connexon formation on the GL membrane was observed by transmission electron microscope. Hydrophilic fluorescent dye transfers were observed through a Cx43‐mediated pathway not only between Sf9 (Spodoptera frugiperda) cells with Cx43 but also from giant Cx43 liposomes to Cx43‐expressing U2OS cells (human osteosarcoma cell). The functional connexin‐containing liposome is expected to be useful for cellular cytosolic delivery systems. The original orientation and function of Cx43 was maintained after integration into the liposomes. The liposome fusion method will create new opportunities as a tool for analysis of channel membrane proteins. Biotechnol. Bioeng. 2010;107: 836–843. © 2010 Wiley Periodicals, Inc.     

A novel subunit of axonemal dynein conserved among lower and higher eukaryotes

To elucidate the subunit composition of axonemal inner-arm dynein, we examined a 38 kDa protein (p38) co-purified with a Chlamydomonas inner arm subspecies, dynein d. We found it is a novel protein conserved among a variety of organisms with motile cilia and flagella. Immunoprecipitation using specific antibody verified its association with a heavy chain, actin and a previously identified light chain (p28). Unexpectedly, mutant axonemes lacking dynein d and other dyneins retained reduced amounts of p38. This finding suggests that p38 is involved in the docking of dynein d to specific loci.     

Recognition of nucleotide analogs containing the 7,8-dihydro-8-oxo structure by the human MTH1 protein

The MTH1 protein catalyzes hydrolysis of oxidatively damaged purine nucleotides including 8-hydroxy-dGTP to the monophosphates. The MTH1 protein seems to act as an important defense system against mutagenesis, carcinogenesis, and cell death induced by oxidized purine nucleotides. We previously reported that the functional groups at the 2- and 6-positions of the purine ring affect the recognition by the human MTH1 protein. 8-Hydroxy-dGTP and 8-hydroxy-dATP are substrates of MTH1, and both have the "7,8-dihydro-8-oxo structure." In this study, three nucleotide analogs containing this motif were examined. A synthetic purine analog containing the 7,8-dihydro-8-oxo structure and the 2-amino function (dJTP) was hydrolyzed to the monophosphate with high efficiency by MTH1. On the other hand, two analogs that lack the two-ring system of their bases [formamidopyrimidine-dGTP (FAPY-dGTP) and 2-OH-dYTP] were poor substrates. FAPY-dGTP is a mixture of conformers and was hydrolyzed more than ten-fold less efficiently than 8-hydroxy-dGTP. These results clarify the effects of the 2-amino group and the two-ring system of the purine base on the recognition by the human MTH1 protein.     

Intranuclear disposition of exogenous DNA in vivo: silencing, methylation and fragmentation

The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed. Expression efficiency from one copy of the exogenous DNA dramatically decreased over time, and the DNA was methylated and degraded into fragments. Unexpectedly, methylation of the intact plasmid DNA was low and did not increase over time. Rather, the fragmented DNA was methylated more frequently than the intact plasmid. These results suggest that the CpG methylation and the degradation of exogenous DNA, and its 'silencing', occurred in parallel in the nucleus.     

Ecological analysis of benthic ostracods in the northern Japan Sea, based on water properties of modern habitats and late Cenozoic fossil records

The ecology of ostracods in the northern Japan Sea is well documented. These ostracods can be grouped in two assemblages: (1) the Tsushima Warm Current Surface Water Assemblage (TWSA, northern), which lives in an environment where summer temperatures reach up to about 20 °C, winter temperatures up to about 5 °C. Several species in this assemblage, e.g., Loxoconcha optima and Pontocythere subjaponica tolerate seasonally low temperatures, and lived in the region since the early Pleistocene; (2) the Japan Sea Intermediate Proper Water Assemblage (JSI-PA), which lives at greater depths (lower shelf to upper slope). This assemblage lives under stable conditions, with a small annual range in temperature (0–5 °C). The assemblage has a relatively low diversity, contains species such as Acanthocythereis dunelmensis and Robertsonites spp., and has been present in the area since the Pliocene. These data indicate that the presently living species either tolerate seasonally low temperatures (TWSA, northern) or tolerate stable, but very low temperatures (near 0 °C), and therefore, they could survive the Pleistocene environmental fluctuations in the Japan Sea caused by glacio-eustatic changes in sea level. Our data document the survival of ostracod species during past climate change, and thus can be used to speculate on the effect of possible future climate change on the faunas. We predict that some of the cryophilic species in the Japan Sea cannot be expected to survive global warming for more than 2 centuries.     

Evaluation of a new character for the phylogenetic analysis of Ostracoda (Crustacea): the podocopan maxillular branchial plate

A data set of the number of setae on the maxillular branchial plate of podocopan ostracods was compiled from published drawings and our own dissections. A total of 168 species in six superfamilies were covered, with the Cytheroidea, Cypridoidea and Bairdioidea being best represented. The number of setae is shown to be independent of the size of the ostracod and the environment in which the species lives, but to have phylogenetic significance at superfamily level. Ontogenetic data suggest that the low number of branchial plate setae in many cytheroidean families is a paedomorphic phenomenon.     

Adaptive regulation of wall shear stress optimizing vascular tree function

The branching structure of the mammalian arterial tree has been known to be close to that of an optimal conduit system of the minimum work model characterized as the branch system of constant wall shear rate. The physiological mechanism producing such construction was considered to be based on the local response of arterial caliber induced by the wall shear stress (shear rate × blood viscosity) and thereby maintaining this stress constant, which was previously observed at the canine common carotid artery shunted to the external jugular vein. The stress levels at various parts of the arterial system estimated from available data fell within ±50% of the mean (15 dyn/cm²), which was consistent with the value predicted from the model. Theoretical analyses on the cost function of the model indicated that the suspected variation of shear rate levels in the arterial tree due to the anomalous changes in blood viscosity which might bring about 3- to 4-fold differences between the minimum and maximum shear rates would cause less than 10% increase in the total energy cost. It was concluded that a local adaptive response to wall shear stress is the mechanism which effectively optimizes the design of the arterial tree.     

Eremophilanolides and other constituents from the Argentine liverwort Frullania brasiliensis.

Two eremophilanolides, 5-epidilatanolides A and B, as well as a new natural bibenzyl were isolated from an Argentine collection of the liverwort Frullania brasiliensis, along with the known eudesmane-type sesquiterpene lactones nepalensolide A, nepalensolide B, (+)-frullanolide, and (+)-dihydrofrullanolide, the hopanoid zeorin, the four sterols stigmasta-4,22-dien-3,6-dione, stigmasta-4,22-dien-3-one, stigmasterol, and sitosterol, and a trace amount of atraric acid. The structure and stereochemistry of the eremophilanolides and the bibenzyl were established by a combination of extensive NMR spectroscopy experiments and X-ray crystallographic analysis. Absolute configurations of the new compounds were derived on the basis of CD spectra.     

A disk-type apparatus for applying fluid shear stress on cultured endothelial cell

To study the effect of fluid shear stress on cultured endothelial cells, we have developed an apparatus for the stress creation, which consists of a stainless steel disk driven by an electric DC motor and a stage to place a culture dish and to adjust the distance between the disk and the dish. When the disk is rotated, a concentric fluid movement occurs in the culture medium in the dish and exerts the shear stress on the endothelial cells cultured on the bottom of the dish. A theoretical analyses concerning the induced concentric flow velocity predicted that when the angular velocity of the disk rotation (omega) is slow enough to maintain a Reynolds' number of the order of 10, the exerted wall shear stress tau w on the endothelial cell monolayer is given for a constant as tau w = mu r omega/d where mu is the viscosity of the medium, d the distance from the plate to the monolayer and r the radial distance from the center of the dish. When omega is varied in a sinusoidal mode tau w also becomes sinusoidal, thus allowing to apply a pulsatile stress. In vitro experiments carried out to examine the validity of the theoretical results, using a suspension of polystyrene as a tracer with the ordinary culture medium and 99% ethanol, revealed excellent agreement of the measured velocity profiles with the predicted ones. The results demonstrated that the present apparatus can create both the steady and pulsatile wall shear stress on the culture cell layer as expected, unless Reynolds' number greatly exceeds the level of 10.