Systematic fabrication of nano-carbonated hydroxyapatite/collagen composites for biomimetic bone grafts

A novel biomimetic self-assembly method was designed to create nano-carbonated hydroxyapatite/collagen (nCHAC) composites by means of incorporating various collagen and carbonate concentrations using solutions such as CaCl(2), H(3)PO(4), and Na(2)CO(3). At a given range of collagen and carbonate content, the nanosized inorganic phase of the newly synthesized material has a low degree of crystallinity which resembles that of natural bone. By manipulating the concentrations of collagen and carbonates, various morphologies of the nCHAC can be obtained. The crystal size of nCHAC is dependent on the concentration of carbonate and collagen present in the composites. For instance, higher collagen concentration results in smaller crystal nCHAC crystal size. Conversely, the higher the carbonate content, the smaller are the crystal size and the collagen fibril assembly. As the carbonate content increased, the plate-like crystals first became needle-like structures, subsequently short needle-like crystals and eventually became spherical particles. From this study, our method showcased the flexibility of fabricating various types of nCHAC composites which can be designed for different bone applications.     

Strict preparation and evaluation of water-soluble hat-stacked carbon nanofibers for biomedical application and their high biocompatibility: influence of nanofiber-surface functional groups on cytotoxicity

Water-soluble H-CNFs modified with a carboxyl group possessed the ability to induce TNF-alpha, whereas CHAPS-treated H-CNFs possessed significantly greater activity and were also found to activate NF-kappaB reporter activity, to a significantly greater level than H-CNFs; furthermore the functional group modified or coated on the surface of H-CNFs was a significant cytotoxic factor that affected cell activation.     

Aromatization of androstenedione and 16alpha-hydroxyandrostenedione in human placental microsomes. Kinetic analysis of inhibition by the 19-oxygenated and 3-deoxy analogs

Inhibition of aromatase activity in human placental microsomes with androstenedione (AD) (1a) and its 19-oxygenated derivatives 1b and 1c, their 16alpha-hydroxy compounds 2 and 3, and 3-deoxyandrost-4-ene compounds 5 and 6 was studied using [1beta-(3)H]AD as a substrate and compared to that with [1beta-(3)H]16alpha-hydroxyandrostenedione (16-OHAD). AD series of steroids, compounds 1, inhibited competitively [1beta-(3)H]AD aromatization whereas other 16alpha-hydroxy steroids 2, 3, 5, and 6 inhibited AD aromatization in a non-competitive manner. On the other hand, all of 16-OHAD series, compounds 2, blocked the [1beta-(3)H]16-OHAD aromatization in a competitive manner whereas the AD series steroids 1 as well as the 3-deoxy-16alpha-hydroxy-17-one steroids 5 and 3-deoxy-16alpha,17beta-diol steroids 6 inhibited 16-OHAD aromatization non-competitively. 3-Carbonyl and 16alpha-hydroxy functions of 16-OHAD play a critical role of selection of the 16-OHAD binding site. The results suggest that the AD derivatives 1 are kinetically aromatized at a different site from the 16-OHAD derivatives 2. Physical and/or chemical environments around the aromatase protein in the microsomal membrane may play a significant role in the expression of the substrate specificity, and the present results do not exclude the idea that the placental microsomes have a single binding site.     

Acidic bile salts modulate the squamous epithelial barrier function by modulating tight junction proteins

Experimental models for esophageal epithelium in vitro either suffer from poor differentiation or complicated culture systems. An air-liquid interface system with normal human bronchial epithelial cells can serve as a model of esophageal-like squamous epithelial cell layers. Here, we explore the influence of bile acids on barrier function and tight junction (TJ) proteins. The cells were treated with taurocholic acid (TCA), glycocholic acid (GCA), or deoxycholic acid (DCA) at different pH values, or with pepsin. Barrier function was measured by transepithelial electrical resistance (TEER) and the diffusion of paracellular tracers (permeability). The expression of TJ proteins, including claudin-1 and claudin-4, was examined by Western blotting of 1% Nonidet P-40-soluble and -insoluble fractions. TCA and GCA dose-dependently decreased TEER and increased paracellular permeability at pH 3 after 1 h. TCA (4 mM) or GCA (4 mM) did not change TEER and permeability at pH 7.4 or pH 4. The combination of TCA and GCA at pH 3 significantly decreased TEER and increased permeability at lower concentrations (2 mM). Pepsin (4 mg/ml, pH 3) did not have any effect on barrier function. DCA significantly decreased the TEER and increased permeability at pH 6, a weakly acidic condition. TCA (4 mM) and GCA (4 mM) significantly decreased the insoluble fractions of claudin-1 and claudin-4 at pH 3. In conclusion, acidic bile salts disrupted the squamous epithelial barrier function partly by modulating the amounts of claudin-1 and claudin-4. These results provide new insights for understanding the role of TJ proteins in esophagitis.     

Multiple functions of G protein-coupled receptor kinases

Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1–GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of β-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of β-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of β-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the β-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and β-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for β-arrestin-mediated signaling. Agonists that selectively activate GRK/β-arrestin-dependent signaling without affecting G protein signaling are known as β-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as signaling mediators that are independent of either G protein- or β-arrestin-mediated pathways. GRKs can phosphorylate non-GPCR substrates, and this is found to be involved in various physiological responses, such as cell motility, development, and inflammation. In addition to these effects, our group revealed that GRK6 expressed in macrophages mediates the removal of apoptotic cells (engulfment) in a kinase activity-dependent manner. These studies revealed that GRKs block excess stimulus and also induce cellular responses. Here, we summarized the involvement of GRKs in β-arrestin-mediated and G protein-independent signaling pathways.     

Effects of exotic mongoose (Herpestes javanicus) on the native fauna of Amami-Oshima Island, southern Japan, estimated by distribution patterns along the historical gradient of mongoose invasion

We examined the distribution patterns of native animals on Amami-Oshima Island, southern Japan, along a historical gradient of mongoose establishment and estimated the effects of mongoose on the native fauna. To assess the relative abundance of various ground-dwelling animals, we used the following four methods; sensor cameras for exotic mammals, nighttime driving census for nocturnal native vertebrates, line census for ground-dwelling lizards, and adhesive traps for arthropods. The results indicated that seven species with larger body size, including mammals, birds, reptiles, and amphibians, were rarely observed in mongoose-infested area. By contrast, medium-sized animals showed neutral relationships with mongoose establishment. Interestingly, the densities of smaller-sized animals were higher in mongoose-infested area. It could be interpreted that smaller species have increased in abundance through top-down cascades, i.e., decreases in native predators such as frogs and lizards caused by the mongoose have resulted in increases in the abundance of smaller animals. Predation pressures by mongoose and native predators may be canceled out for medium-sized animals, causing neutral responses to mongoose by these animals. This study appears to be the first example that shows the influence of mongoose on a wide variety of native animals. In addition, our findings indicate the importance of considering the food web structure of a recipient ecosystem and contribute to the prediction and assessment of ecological risks caused not only by mongoose, but also by other invasive top predators.     

In vivo 31P MRS in new antineoplastic agents evaluation on experimental tumor models

Two new antineoplastic agents, a nitrosourea and a DNA-bis-intercalator have been studied in vivo by 31P magnetic resonance spectroscopy on a rat glioma and Walker carcinoma. On rat glioma, spectra are modified when the tumor is treated by the nitrosourea, showing the depletion of high-energy phosphates. On Walker carcinoma both drugs delay the tumor evolution to necrosis, showing important levels of high-energy phosphates on NMR spectra. There appears to be a great dependence upon energy metabolism during chemotherapy, depending on the nature and physiology of the observed tumor.