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.     

Localization of the ultraviolet-sensor Opn5m and its effect on myopia-related gene expression in the late-embryonic chick eye

Recent studies show that exposure to ultraviolet (UV) light suppresses ocular elongation, which causes myopia development. However, the specific mechanisms of this process have not been elucidated. A UV-sensor, Opsin 5 (Opn5) mRNA was shown to be present in extraretinal tissues. To test the possibility that UV-signals mediated by Opn5 would have a direct effect on the outer connective tissues of the eye, we first examined the expression patterns of a mammalian type Opn5 (Opn5m) in the late-embryonic chicken eye. Quantitative PCR showed Opn5m mRNA expression in the cornea and sclera. The anti-Opn5m antibody stained a small subset of cells in the corneal stroma and fibrous sclera. We next assessed the effect of UV-A (375 nm) irradiation on the chicken fibroblast cell line DF-1 overexpressing chicken Opn5m. UV-A irradiation for 30 min significantly increased the expression of Early growth response 1 (Egr1), known as an immediate early responsive gene, and of Matrix metalloproteinase 2 (Mmp2) in the presence of retinal chromophore 11-cis-retinal. In contrast, expression of Transforming growth factor beta 2 and Tissue inhibitor of metalloproteinase 2 was not significantly altered. These results indicate that UV-A absorption by Opn5m can upregulate the expression levels of Egr1 and Mmp2 in non-neuronal, fibroblasts. Taken together with the presence of Opn5m in the cornea and sclera, it is suggested that UV-A signaling mediated by Opn5 in the extraretinal ocular tissues could influence directly the outer connective tissues of the chicken late-embryonic eye.     

Serotonin-immunoreactive neurons in the antennal sensory system of the brain in the carpenter ant, Camponotus japonicus

Social Hymenoptera such as ants or honeybees are known for their extensive behavioral repertories and plasticity. Neurons containing biogenic amines appear to play a major role in controlling behavioral plasticity in these insects. Here we describe the morphology of prominent serotonin-immunoreactive neurons of the antennal sensory system in the brain of an ant, Camponotus japonicus. Immunoreactive fibers were distributed throughout the brain and the subesophageal ganglion (SOG). The complete profile of a calycal input neuron was identified. The soma and dendritic elements are contralaterally located in the lateral protocerebrum. The neuron supplies varicose axon terminals in the lip regions of the calyces of the mushroom body, axon collaterals in the basal ring but not in the collar region, and other axon terminals ipsilaterally in the lateral protocerebrum. A giant neuron innervating the antennal lobe has varicose axon terminals in most of 300 glomeruli in the ventral region of the antennal lobe (AL) and a thick neurite that spans the entire SOG and continues towards the thoracic ganglia. However, neither a soma nor a dendritic element of this neuron was found in the brain or the SOG. A deutocerebral projection neuron has a soma in the lateral cell-body group of the AL, neuronal branches at most of the 12 glomeruli in the dorsocentral region of the ipsilateral AL, and varicose terminal arborizations in both hemispheres of the protocerebrum. Based on the present results, tentative subdivisions in neuropils related to the antennal sensory system of the ant brain are discussed.     

Crystal structures of two hemoglobin components from the midge larva Propsilocerus akamusi (Orthocladiinae, Diptera)

The polymorphic components of hemoglobin (Hb) of the midge larva Propsilocerus akamusi were classified into two distinct types dependent on their spectroscopic properties, normal absorption (N) and low absorption (L). Analyses of the amino acid sequences of component VII (N-type Hb) and component V (L-type Hb) from P. akamusi indicated that one remarkable difference is the replacement of the distal histidine (His) with isoleucine (Ile) in component V. To clarify the structural differences between the two Hb components, we determined the crystal structures of components V and VII at resolutions of 1.64 A and 1.50 A, respectively. These crystal structures indicated a short additional helix comprising three amino acid residues at the C-terminal region in component V, and a typical globin fold including eight helices in component VII. Comparison of the heme regions of the Hb components suggests that the structural changes of the heme region in component V on ligation differ from that of usual Hb.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of a low glutelin mutant of ‘Koshihikari’ rice variety using the mutated-acetolactate synthase gene derived from rice genome as a selectable marker

We have developed an efficient rice transformation system that uses only rice genome-derived components. The transgenic ‘Koshihikari’ rice, low-glutelin mutant a123, is capable of accumulating large amounts of bioactive peptides in the endosperm. Agrobacterium-mediated transformation using the mutated-acetolactate synthase (mALS) gene expressed under the control of the callus-specific promoter (CSP) as a selectable marker was used to introduce GFP and an anti-hypertensive hexapeptide into ‘Koshihikari’ a123. The CSP:mALS gene cassette confers pyrimidinyl carboxy herbicide resistance to transgenic rice callus, but is not expressed in regenerated plants. Transformation efficiency of transgenic rice line a123 was improved from about 10% to about 30% by modifying callus induction, callus selection and regeneration media conventionally used for rice tissue culture. An erratum to this article can be found at     

Metabolic flux analysis in plants using dynamic labeling technique: application to tryptophan biosynthesis in cultured rice cells

The concept and methodology of using dynamic labeling for the MFA of plant metabolic pathways are described, based on a case study to develop a method for the MFA of the tryptophan biosynthetic pathway in cultured rice cells. Dynamic labeling traces the change in the labeling level of a metabolite in a metabolic pathway after the application of a stable isotope-labeled compound. In this study, [1-(13)C] l-serine was fed as a labeling precursor and the labeling level of Trp was determined by using the LC-MS/MS. The value of metabolic flux is determined by fitting a model describing the labeling dynamics of the pathway to the observed labeling data. The biosynthetic flux of Trp in rice suspension cultured cell was determined to be 6.0+/-1.1 nmol (gFWh)(-1). It is also demonstrated that an approximately sixfold increase in the biosynthetic flux of Trp in transgenic rice cells expressing the feedback-insensitive version of anthranilate synthase alpha-subunit gene (OASA1D) resulted in a 45-fold increase in the level of Trp. In this article, the basic workflow for the experiment is introduced and the details of the actual experimental procedures are explained. Future perspectives are also discussed by referring recent advances in the dynamic labeling approach.     

Expression of murine killer immunoglobulin-like receptor KIRL1 on CD1d-independent NK1.1<Superscript>+</Superscript> T cells

Three mouse killer immunoglobulin-like receptors (KIRs), namely, KIR3DL1, KIRL1, and KIRL2, have recently been identified in C56BL/6 (B6) mice. However, only two Kir genes are found in the B6 mouse genome sequence data base. To clarify this discrepancy, we cloned Kir cDNAs from multiple strains of mice. Sequencing of the cDNA clones showed that the Kir3dl1 gene is found in C3H/HeJ and CBA/J but not in B6 mice. Analysis of the single nucleotide polymorphism data base suggested that Kir3dl1 is the C3H/HeJ and CBA/J allele of Kirl1. We generated mAb to the recombinant KIRL1 protein to investigate its expression pattern. The anti-KIRL1 mAb bound to NK1.1⁺ T cells but only very weakly or at undetectable levels to other lymphocytes including natural killer (NK) cells and conventional T cells. Among NK1.1⁺ T cells, conventional NK T cells stained with CD1d tetramer did not significantly bind anti-KIRL1 mAb, whereas CD1d-tetramer-negative subset was KIRL1-positive. Furthermore, the expression of KIRL1 is readily detected on NK1.1⁺ T cells from β₂-microglobulin-deficient B6 mice. Thus, KIRL1 is predominantly expressed on CD1d-independent NK1.1⁺ T cells.     

Abundance decline in the narrow-ridged finless porpoise population off the Pacific coast of eastern Japan

The narrow-ridged finless porpoise Neophocaena asiaeorientalis is a small-toothed whale with a coastal habitat and is sensitive to human activity. Aerial sighting surveys were conducted in 2012 to evaluate the abundance and distribution of the Sendai Bay-Tokyo Bay population off the Pacific coast of eastern Japan. We flew along 35 east–west transects at intervals of 11.6 km over the study area between 34°57′ and 38°21’ N. In total, 25 groups were detected by two observers. The mean group size was 1.44 individuals. Porpoise abundance was estimated to be 1,491 individuals (coefficient of variation = 32.4%), which was lower than the abundance estimated in 2000 (z test, p < 0.05). A population decline trend during 2000–2012 was also detected using four available abundance estimates (Bootstrapping linear regression, p = 0.040). A possible cause of this decline was coastal habitat disturbance and deaths by tsunami waves caused by the 2011 Great East Japan Earthquake. Density was as low as 0.221 individuals/km², which is among the lowest levels in Japanese waters. Furthermore, distributional gaps reconfirmed by the present study and past genetic studies show that the population should be divided into at least two populations: northern (“Sendai Bay-Fukushima”) and southern. Density decline was detected in the northern population between 2000 and 2012. Unlike the earthquake, human activities may continue to affect the porpoises. Viability analyses of these small populations should be conducted in the future.     

Aurora-A Breaks Symmetry in Contractile Actomyosin Networks Independently of Its Role in Centrosome Maturation

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