The differential amino acid requirement within osteopontin in α4 and α9 integrin-mediated cell binding and migration

Osteopontin (OPN) contains at least two major integrin recognition domains, Arg159-Gly-Asp161 (RGD) and Ser162-Val-Val-Tyr-Gly-Leu-Arg168 (SVVYGLR), recognized by αvβ3 and α5β1 and α4 and α9 integrins, respectively. OPN is specifically cleaved by thrombin and matrix metalloproteinase (MMP)-3 or MMP-7 at a position of Arg168/Ser169 (R/S) and Gly166/Leu167 (G/L), respectively. We in this study examined the requirement of residues within SVVYGLR for the α4 and α9 integrin recognition and how MMP-cleavage influences the integrin recognition. The residues, Val164, Tyr165, and Leu167 are critical for α4 and α9 integrin recognition in both cell adhesion and cell migration. The residue Arg168 is additionally required for α9 integrin recognition in cell adhesion and this explains why α9 integrin binds to only thrombin cleaved form of OPN. α4 integrin is able to bind to SVVYG (MMP-cleaved form of RAA OPN-N half), while α9 integrin is not, supporting the above notion that Arg168 is additionally required for α9 integrin-mediated cell adhesion. The residue Val163 is important for α4, but not for α9 integrin recognition in cell migration. Importantly, we found that the replacement of Arg168 by Ala (R168A mutant) induces the augmentation of cell migration via α4 and α9 integrins.     

Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals

Here, we demonstrate self-organized formation of apico-basally polarized cortical tissues from ESCs using an efficient three-dimensional aggregation culture (SFEBq culture). The generated cortical neurons are functional, transplantable, and capable of forming proper long-range connections in vivo and in vitro. The regional identity of the generated pallial tissues can be selectively controlled (into olfactory bulb, rostral and caudal cortices, hem, and choroid plexus) by secreted patterning factors such as Fgf, Wnt, and BMP. In addition, the in vivo-mimicking birth order of distinct cortical neurons permits the selective generation of particular layer-specific neurons by timed induction of cell-cycle exit. Importantly, cortical tissues generated from mouse and human ESCs form a self-organized structure that includes four distinct zones (ventricular, early and late cortical-plate, and Cajal-Retzius cell zones) along the apico-basal direction. Thus, spatial and temporal aspects of early corticogenesis are recapitulated and can be manipulated in this ESC culture.     

A novel recombinant system for functional expression of myonecrotic snake phospholipase A(2) in Escherichia coli using a new fusion affinity tag

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A₂ ([Lys⁴⁹]PLA₂), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as α-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for α-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity.     

Controlling the direction of kinesin-driven microtubule movements along microlithographic tracks

Motor proteins are able to move protein filaments in vitro. However, useful work cannot be extracted from the existing in vitro systems because filament motions are in random directions on two-dimensional surfaces. We succeeded in restricting kinesin-driven movements of microtubules along linear tracks by using micrometer-scaled grooves lithographically fabricated on glass surfaces. We also accomplished the extraction of unidirectional movement from the bidirectional movements along the linear tracks by adding arrowhead patterns on the tracks. These "rectifiers" enabled us to construct microminiturized circulators in which populations of microtubules rotated in one direction, and to actively transport microtubules between two pools connected by arrowheaded tracks in the fields of micrometer scales.     

Calcium-induced changes in chondroitin sulfate chains of urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI) has several roles other than protease inhibition. It is suggested that UTI inhibits calcium influx in cultured cells and that the chondroitin sulfate chain of UTI may play an important role. In order to clarify the mechanistic features of this phenomenon, the chondroitin sulfate chain of UTI was analyzed by (1)H-NMR. The samples were highly purified UTI dissolved in D(2)O in the presence or absence of Ca(2+), Mg(2+) and Na(+). 1D-spectra were obtained and T(1) values of detected signals were estimated from the inversion-recovery method. The addition of Ca(2+) to UTI caused a chemical shift to downfield, line broadening and a reduction of T(1) values at several signals from chondroitin sulfate moiety (especially at axial H-2 of GalNAc), whereas Mg(2+) and Na(+) had no significant effect. Some of the signals in the linkage region of chondroitin sulfate chain showed marked line broadening by Ca(2+). The reduction of T(1) values implies formation of a complex. It is suggested that Ca(2+) generates the sulfate salt and interacts with other polar groups in the chondroitin sulfate chain, thereby causing bridging between UTI molecules. Several properties of UTI may be related to this interaction of Ca(2+) with chondroitin sulfate chains.     

Bryophyte 5S rDNA was inserted into 45S rDNA repeat units after the divergence from higher land plants

The 5S ribosomal RNA genes (5S rDNA) are located independently from the 45S rDNA repeats containing 18S, 5.8S and 26S ribosomal RNA genes in higher eukaryotes. Southern blot and fluorescence in situ hybridization analyses demonstrated that the 5S rDNAs are encoded in the 45S rDNA repeat unit of a liverwort, Marchantia polymorpha, in contrast to higher plants. Sequencing analyses revealed that a single-repeat unit of the M. polymorpha nuclear rDNA, which is 16103 bp in length, contained a 5S rDNA downstream of 18S, 5.8S and 26S rDNA. To our knowledge, this is the first report on co-localization of the 5S and 45S rDNAs in the rDNA repeat of land plants. Furthermore, we detected a 5S rDNA in the rDNA repeat of a moss, Funaria hygrometrica, by a homology search in a database. These findings suggest that there has been structural re-organization of the rDNAs after divergence of the bryophytes from the other plant species in the course of evolution.     

Morphological analyses of interleukin-8 effects on rat ovarian follicles at ovulation and luteinization in vivo

The aim of the present study was to elucidate functions of the interleukin (IL)-8 at ovulation and luteinization in vivo. To compare the morphological differences between human chorionic gonadotropin (hCG) and IL-8 stimulation, scanning electron microscopy was employed to study rat ovarian vascular corrosion casts. Follicular growth and increased capillary vessel densities around the follicles were seen in vascular corrosion casts after IL-8 injection, similar to the result of hCG administration. This result indicated that exogenous IL-8 could play a role in the neovascularization during follicular development as an angiogenetic factor. Many fenestrations were observed in the vascular endothelium by hCG administration. In contrast, no fenestrations were observed with IL-8 injection, indicating that IL-8 may not be sufficient to increase the vascular permeability directly. Although germinal vesicle breakdown (GVBD) occurred at rates of 82% after the hCG injection, only 20% GVBD was observed after the IL-8 injection. The present study indicated that IL-8 might have important effects on rat follicles at ovulation and luteinization via vascularization in a similar manner to hCG. However, IL-8 was not effective on vascular permeability and oocyte maturation, which were different from hCG. Thus, we can conclude that IL-8 can participate in follicular development in part and may play important roles in ovulation and luteinization as one of some mediators induced by endogenous luteinizing hormone.