Generation of knock-in mice carrying third cones with spectral sensitivity different from S and L cones

Red-green color vision in primates is unique in the sense that it is mediated by two photoreceptor cells that are indistinguishable in all aspects except for their visual pigments. In order to generate an animal model for investigation of the interaction between red-green inputs at the molecular level, we applied knock-in technology and X-chromosome inactivation machinery to make a mouse model with cone cells possessing visual pigments with different spectral sensitivities. We introduced a S308A point mutation into the Green opsin gene allele on the X-chromosome. This manipulation generated a 24 nm red-shift of absorption maximum in the cone pigment with negligible functional differences in other molecular properties. Amplitudes of responses in ERG and ganglion cell recordings of homozygotes were similar to those of wild-types, although the spectral sensitivities differed. Heterozygotes showed variable spectral sensitivities of ganglion cell responses due to the different integration of the native and the S308A cone inputs on the dendritic fields. In situ hybridization experiments showed that cone cells with respective pigments formed patch-like clusters of specific L cone-types, approximately 30 mum in diameter, which were randomly distributed in the dorsal region of the retinas. Since the patch-like clustering was arranged by X-inactivation, such clustering could be present in the peripheral retinas of New World monkeys with polymorphic L pigments, indicating that our mice would be a suitable model to study evolution of the mammalian color vision system.     

Purification and characterization of a feruloyl esterase from the intestinal bacterium Lactobacillus acidophilus

Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37 degrees C, respectively). The metal ions Cu(2+) and Fe(3+) (at a concentration of 5 mmol liter(-1)) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-L-arabinofuranose (FAA) as a substrate, the enzyme exhibited a K(m) of 0.0953 mmol liter(-1) and a V(max) of 86.27 mmol liter(-1) min(-1) mg(-1) of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA from O-(5-O-feruloyl-alpha-L-arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase.     

Physiological properties of rod photoreceptor cells in green-sensitive cone pigment knock-in mice

Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was approximately 11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 x 10(-7) s(-1), about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.     

A Critical Interpersonal Distance Switches between Two Coordination Modes in Kendo Matches

In many competitive sports, players need to quickly and continuously execute movements that co-adapt to various movements executed by their opponents and physical objects. In a martial art such as kendo, players must be able to skillfully change interpersonal distance in order to win. However, very little information about the task and expertise properties of the maneuvers affecting interpersonal distance is available. This study investigated behavioral dynamics underlying opponent tasks by analyzing changes in interpersonal distance made by expert players in kendo matches. Analysis of preferred interpersonal distances indicated that players tended to step toward and away from their opponents based on two distances. The most preferred distance enabled the players to execute both striking and defensive movements immediately. The relative phase analysis of the velocities at which players executed steps toward and away revealed that players developed anti-phase synchronizations at near distances to maintain safe distances from their opponents. Alternatively, players shifted to in-phase synchronization to approach their opponents from far distances. This abrupt phase-transition phenomenon constitutes a characteristic bifurcation dynamics that regularly and instantaneously occurs between in- and anti-phase synchronizations at a critical interpersonal distance. These dynamics are profoundly affected by the task constraints of kendo and the physical constraints of the players. Thus, the current study identifies the clear behavioral dynamics that emerge in a sport setting.     

Gene trapping in Arabidopsis reveals genes involved in vascular development

The procambium is made up of stem cells that give rise to various vascular cells in plants. To understand the molecular nature of procambium cells, we tried to identify genes that characterize procambium cells using Arabidopsis gene trap lines. Among 26,000 gene trap lines, we found 67 lines in which beta-glucuronidase (GUS) staining occurred along vascular tissues in cotyledons and/or adult leaves. Although four gene trap lines showed procambium-preferential GUS expression, their expression patterns differed from each other during procambium development in root tips and young rosette leaves. Genomic regions flanking the gene trap insertion points in 25 of the 67 lines were determined, including three lines showing preferential GUS staining of the procambium. The three procambium-related genes encoded PINHEAD, katanin and an unknown DUF740 domain-containing protein. We discuss procambium development based on the functions and the differential GUS staining patterns of the procambium-related genes.     

Expression of endothelia and lymphocyte adhesion molecules in bronchus-associated lymphoid tissue (BALT) in adult human lung

Background

The aim of the present study was to explore the occurrence of fibrocytes in tissue and to investigate whether the appearance of fibrocytes may be linked to structural changes of the parenchyme and vasculature in the lungs of patients with obliterative bronchiolitis (OB) following lung or bone marrow transplantation.

Methods

Identification of parenchyme, vasculature, and fibrocytes was done by histological methods in lung tissue from bone marrow or lung-transplanted patients with obliterative bronchiolitis, and from controls.

Results

The transplanted patients had significantly higher amounts of tissue in the alveolar parenchyme (46.5 ± 17.6%) than the controls (21.7 ± 7.6%) (p < 0.05). The patients also had significantly increased numbers of fibrocytes identified by CXCR4/prolyl4-hydroxylase, CD45R0/prolyl4-hydroxylase, and CD34/prolyl4-hydroxylase compared to the controls (p < 0.01). There was a correlation between the number of fibrocytes and the area of alveolar parenchyma; CXCR4/prolyl 4-hydroxylase (p < 0.01), CD45R0/prolyl 4-hydroxylase (p < 0.05) and CD34/prolyl 4-hydroxylase (p < 0.05). In the pulmonary vessels, there was an increase in the endothelial layer in patients (0.31 ± 0.13%) relative to the controls (0.037 ± 0.02%) (p < 0.01). There was a significant correlation between the number of fibrocytes and the total area of the endothelial layer CXCR4/prolyl 4-hydroxylase (p < 0.001), CD45R0/prolyl 4-hydroxylase (p < 0.001) and CD34/prolyl 4-hydroxylase (p < 0.01). The percent areas of the lumen of the vessels were significant (p < 0.001) enlarged in the patient with OB compared to the controls. There was also a correlation between total area of the lumen and number of fibrocytes, CXCR4/prolyl 4-hydroxylase (p < 0.01), CD45R0/prolyl 4-hydroxylase (p < 0.001) and CD34/prolyl 4-hydroxylase (p < 0.01).

Conclusion

Our results indicate that fibrocytes are associated with pathological remodelling processes in patients with OB and that tissue fibrocytes might be a useful biomarker in these processes.     

Transgenic expression of osteoactivin in the liver attenuates hepatic fibrosis in rats

The role of osteoactivin (OA) in liver fibrogenesis remains unclear. After feeding wild-type (WT) and OA transgenic (OA-Tg) rats a choline-deficient, L-amino acid-defined (CDAA) diet for 12 weeks, we evaluated liver fibrosis. Hepatic fibrosis and expression of alpha-smooth muscle actin protein in OA-Tg rats were reduced in comparison to WT rats. Our examination of the expression of 31,100 genes by microarray analysis identified 177 and 256 genes that were upregulated and downregulated, respectively, by at least twofold in OA-Tg rat livers in comparison to WT rat livers. Of these genes, we confirmed a significant downregulation in the expression levels of tissue inhibitor of metalloproteinase-1 and -2, type I collagen, and platelet-derived growth factor receptor-alpha and -beta in the livers of OA-Tg rats. These results indicate that transgenic OA expression attenuates the development of hepatic fibrosis in association with the suppression of specific genes involved in its pathogenesis.