Co-ordinated ocular development from human iPS cells and recovery of corneal function.

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and collagen-rich stroma of the cornea have a neural crest origin. Recent work with pluripotent stem cells in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. Moreover, we and others have demonstrated the in vitro induction of a corneal epithelial cell phenotype from pluripotent stem cells. These studies, however, have a single, tissue-specific focus and fail to reflect the complexity of whole eye development. Here we demonstrate the generation from human induced pluripotent stem cells of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. In some respects the concentric SEAM mimics whole-eye development because cell location within different zones is indicative of lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. It thus represents a promising resource for new and ongoing studies of ocular morphogenesis. The approach also has translational potential and to illustrate this we show that cells isolated from the ocular surface ectodermal zone of the SEAM can be sorted and expanded ex vivo to form a corneal epithelium that recovers function in an experimentally induced animal model of corneal blindness.

Three-dimensional sensing of surfaces by projection of invisible electroluminescence from organic light-emitting diodes.

Organic light-emitting diodes (OLEDs) that efficiently emit near-infrared (NIR) light and consume little power will create valuable applications for OLEDs beyond just displays. Here, we report such a NIR-OLED with high operational stability that can be used as a light source for three-dimensional sensing of object's surfaces. Using a narrow-energy-gap material as a host for producing NIR hyperfluorescence system, we fabricated a NIR-OLED exhibiting intense emission at 930 nm with a high external electroluminescence quantum efficiency of more than 1% at a current density of 100 milliamperes per square meter without any degradation even after more than 300 hours of operation. The NIR-OLEDs were integrated with dense complementary metal-oxide semiconductor circuits to make a micro-NIR-OLED projector (0.21 inch, 230,400 pixels). By actively driving the projector on a pixel by pixel and projecting their emission onto objects, we successfully scanned and sensed the surfaces in three dimensions with invisible NIR.

Human serum albumin as an antioxidant in the oxidation of (-)-epigallocatechin gallate: participation of reversible covalent binding for interaction and stabilization.

Human serum albumin (HSA) contributes to the stabilization of (-)-epigallocatechin gallate (EGCg) in serum. We characterize in the present study the mechanisms for preventing EGCg oxidation by HSA. EGCg was stable in human serum or buffers with HSA, but (-)-epigallocatechin (EGC) was unstable. We show by comparing EGCg and EGC in a neutral buffer that EGCg had a higher binding affinity than EGC. This indicates that the galloyl moiety participated in the interaction of EGCg with HSA and that this interaction was of critical importance in preventing EGCg oxidation. The binding affinity of EGCg for HSA and protein carbonyl formation in HSA were enhanced in an alkaline buffer. These results suggest the reversible covalent modification of EGCg via Schiff-base formation, and that the immobilization of EGCg to HSA, through the formation of a stable complex, prevented the polymerization and decomposition of EGCg in human serum.

Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin.

Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.

Molecular characterization and significance of phosphoenolpyruvate carboxykinase in a ruminal bacterium, Streptococcus bovis.

To gain knowledge about the significance of phosphoenolpyruvate (PEP) carboxykinase (PCK) in Streptococcus bovis, the sequence of the gene encoding PCK (pck) was determined. Transcriptional analysis indicated that the pck is transcribed in a monocistronic fashion. The level of pck-mRNA was higher when cells were grown on lactose than on glucose, suggesting that PCK synthesis increases when the growth rate is low. The pck-mRNA level was higher in a mutant lacking ccpA, which encodes the catabolite control protein A (CcpA), than in the parent strain, suggesting that pck transcription is suppressed by CcpA. S. bovis PCK showed oxaloacetate (OAA)-decarboxylating activity, but no PEP-carboxylating activity (reverse reaction). In S. bovis, OAA was speculated to be produced from PEP via pyruvate. Disruption of pck in S. bovis resulted in decreased growth rate and cell yield. When a pck-disrupted mutant was grown in a medium lacking amino acids, the lag phase was longer and the cell yield was lower than the case of the parent strain. These results suggest that pck is involved in the initiation of growth, including the induction of amino acid synthesis and energy metabolism.

Involvement of two-component signal transduction system, ComDE, in the regulation of growth and genetic transformation, in the ruminal bacterium Streptococcus bovis.

In ruminants, Streptococcus bovis is considered to be associated with acute rumen acidosis. To elucidate the regulatory mechanisms of S. bovis growth, we investigated the function of the two components of the peptide pheromone-signaling system, ComD and ComE, which are encoded by comD and comE, respectively, via the competence-stimulating peptide ComC, which is encoded by comC. Deletion of entire comC and two-thirds of comD resulted in decreased growth rate, which may be related to the change in the expression of several proteins, as shown by two-dimensional gel electrophoresis. The transcript level of comED was decreased by the disruption of comCD, suggesting that the transcription of comED might be stimulated by ComC. The transformation frequency was decreased by the disruption of comCD. Addition of recombinant ComC to S. bovis cultures increased the growth rate and transformation frequency. In the cultures of mixed ruminal microbes, addition of mature ComC peptide increased the number of S. bovis per total bacterial counts as estimated by the cDNA amounts of 16SrRNA. Thus, the peptide pheromone-signaling system via ComC, D, and E might be involved in the control of S. bovis growth in addition to competence development. This is the first report suggesting that an autoinducing peptide functions in the ruminal ecosystem.

Hepatic stellate cell damage may lead to decreased plasma ADAMTS13 activity in rats.

ADAMTS13 is gaining attention, because its deficiency causes thrombotic thrombocytopenic purpura. Although its regulatory mechanism is not fully understood, we wondered if hepatic stellate cells (HSCs) play a role, because ADAMTS13 mRNA is exclusively expressed in the liver and primarily in HSCs. Plasma ADAMTS13 activity was markedly reduced in dimethylnitrosamine-treated rats, where HSC apoptosis is an essential event, but not in carbon tetrachloride- or thioacetamide-treated rats without HSC apoptosis. Furthermore, plasma ADAMTS13 activity was also reduced in 70% hepatectomized rats, where HSC loss occurs. These results suggest that HSC may be involved in the regulation of plasma ADAMTS13 activity.

Influence of the galloyl moiety in tea catechins on binding affinity for human serum albumin.

The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

Binding affinity of tea catechins for HSA: characterization by high-performance affinity chromatography with immobilized albumin column.

Catechins are the major polyphenols in green tea leaves. Recent studies have suggested that the catechins form complexes with HSA for transport in human blood, and their binding affinity for albumin is believed to modulate their bioavailability. In this study, the binding affinities of catechins and their analogs were evaluated and the relationship between the chemical structure of each catechin and its binding property were investigated. Comparing these catechins by HPLC analysis with the HSA column, we showed that galloylated catechins have higher binding affinities with HSA than non-galloylated catechins. In addition, pyrogallol-type catechins have a high affinity compared to catechol-type catechins. Furthermore, the binding affinity of the catechin with 2,3-trans structure was higher than those of the catechin with 2,3-cis structure. The importance of the hydroxyl group on the galloyl group and B-ring was confirmed using methylated catechins. These results indicate that the most important structural element contributing to HSA binding of tea catechins is the galloyl group, followed by the number of hydroxyl groups on the B-ring and the galloyl group or the configuration at C-2. Our findings provide fundamental information on the relationship between the chemical structure of tea catechins and its biological activity.

Albumin stabilizes (-)-epigallocatechin gallate in human serum: binding capacity and antioxidant property.

(-)-Epigallocatechin gallate (EGCg) is the major component of green tea and is known to show strong biological activity, although it can be easily oxidized under physiological conditions. In this study, we indicate that EGCg is stable in human serum and that human serum albumin (HSA) stabilizes EGCg under aerobic condition. Although EGCg is usually decomposed within 1 h in aqueous solution at neutral pH, EGCg in serum and phosphate buffer (pH 7.4) containing HSA was stable over 1 h, even at neutral and slightly alkaline pH. Under these conditions, EGCg binds to HSA non-covalently. The sulfhydryl group acts as an antioxidant for EGCg oxidation. Incubation of EGCg with HSA is accompanied by the oxidation of a free sulfhydryl group in HSA. These results suggest that the antioxidant property and the binding capacity of HSA contribute to the stabilization of EGCg in human serum.

Localized expression of histamine H1 receptors in syncytiotrophoblast cells of human placenta.

The previous Northern blot analysis and in situ hybridization studies showed that histamine H1-receptor (H1R) mRNA is expressed in human placenta and suggested that H(1)R plays some roles in the function of placenta in pregnancy. To investigate further, it is essential to show the precise location of H1R in the placenta. In the present study, we investigated H1R expression in human placenta by radioligand binding assay and immunohistochemical study using an antibody against human H1R. Placentas were obtained from normal uncomplicated deliveries. Membranes prepared from the tissue exhibited saturable [3H]mepyramine binding (K(d) = 4.0 +/- 0.6 nM and B(max) = 91.4 +/- 4.9 fmol/mg of protein). Stereoisomers of chlorpheniramine inhibited [(3)H]mepyramine binding; d-chlorpheniramine inhibited more potently than l-chlorpheniramine, K(i) values being 1.1 +/- 0.4 and 270 +/- 170 nM, respectively. The placenta tissues were positively immunostained with anti-H1R antibody only in the region of the syncytiotrophoblast of chorionic villus. The tissues were double stained with anti-H1R antibody and an antibody against human chorionic gonadotoropin (hCG) that is solely expressed in placental syncytiotrophoblast cells. The results showed that H1R and hCG were expressed on the same cells, that is, syncytiotrophoblast cells. These results indicate that H1Rs are specifically expressed in syncytiotrophoblast cells of human placenta organ.