Osteopontin attenuates aging-associated phenotypes of hematopoietic stem cells

Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age-related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long-term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin-cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma-derived OPN for HSC aging and identify thrombin-cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging.     

Vacuum-ultraviolet photochemically initiated modification of polystyrene surfaces: morphological changes and mechanistic investigations.

Vacuum-ultraviolet (VUV) irradiation (lambda(exc): 172 +/- 12 nm) of polystyrene films in the presence of oxygen produced not only oxidatively functionalized surfaces, but generated also morphological changes. Whereas OH- and C[double bond, length as m-dash]O-functionalized surfaces might be used for e.g. secondary functionalization, enhanced aggregation or printing, processes leading to morphological changes open new possibilities of microstructurization. Series of experiments made under different experimental conditions brought evidence of two different reaction pathways: introduction of OH- and C[double bond, length as m-dash]O-groups at the polystyrene pathways is mainly due to the reaction of reactive oxygen species (hydroxyl radicals, atomic oxygen, ozone) produced in the gas phase between the VUV-radiation source and the substrate. However, oxidative fragmentation leading to morphological changes, oxidation products of low molecular weight and eventually to mineralization of the organic substrate is initiated by electronic excitation of the polymer leading to C-C-bond homolysis and to a complex oxidation manifold after trapping of the C-centred radicals by molecular oxygen. The pathways of oxidative functionalization or fragmentation could be differentiated by FTIR-ATR analysis of irradiated polystyrene surfaces before and after washing with acetonitrile and microscopic fluorescence analysis of the surfaces secondarily functionalized with the N,N,N-tridodecyl-triaza-triangulenium (TATA) cation. Ozonization of the polystyrene leads to oxidative functionalization of the polymer surface but cannot initiate the fragmentation of the polymer backbone. Oxidative fragmentation is initiated by electronic excitation of the polymer (contact-mode AFM analysis), and evidence of the generation of intermediate C-centred radicals is given e.g. by experiments in the absence of oxygen leading to cross-linking (solubility effects, optical microscopy, friction-mode AFM) and disproportionation (fluorescence).     

Biosynthesis,accumulation and emission of carotenoids, α-tocopherol,plastoquinone, and isoprene in leaves under high photosynthetic irradiance

The localization of isoprenoid lipids in chloroplasts, the accumulation of particular isoprenoids under high irradiance conditions, and channelling of photosynthetically fixed carbon into plastidic thylakoid isoprenoids, volatile isoprenoids, and cytosolic sterols are reviewed. During leaf and chloroplast development in spring plastidic isoprenoid biosynthesis provides primarily thylakoid carotenoids, the phytyl side-chain of chlorophylls and the electron carriers phylloquinone K1, alpha-tocoquinone and alpha-tocopherol, as well as the nona-prenyl side-chain of plastoquinone-9. Under high irradiance, plants develop sun leaves and high light (HL) leaves with sun-type chloroplasts that possess, besides higher photosynthetic CO2 assimilation rates, different quantitative levels of pigments and prenylquinones as compared to shade leaves and low light (LL) leaves. After completion of chloroplast thylakoid synthesis plastidic isoprenoid biosynthesis continues at high irradiance conditions, constantly accumulating alpha-tocopherol (alpha-T) and the reduced form of plastoquinone-9 (PQ-9H2) deposited in the steadily enlarging osmiophilic plastoglobuli, the lipid reservoir of the chloroplast stroma. In sun leaves of beech (Fagus) and in 3-year-old sunlit Ficus leaves the level of alpha-T and PQ-9 can exceed that of chlorophyll b. Most plants respond to HL conditions (sun leaves, leaves suddenly lit by the sun) with a 1.4-2-fold increase of xanthophyll cycle carotenoids (violaxanthin, zeaxanthin, neoxanthin), an enhanced operation of the xanthophyll cycle and an increase of beta-carotene levels. This is documented by significantly lower values for the weight ratio chlorophylls to carotenoids (range: 3.6-4.6) as compared to shade and LL leaves (range: 4.8-7.0). Many plant leaves emit under HL and high temperature conditions at high rates the volatile compounds isoprene (broadleaf trees) or methylbutenol (American ponderosa pines), both of which are formed via the plastidic 1-deoxy-D: -xylulose-phosphate/2-C-methylerythritol 5-phosphate (DOXP/MEP) pathway. Other plants by contrast, accumulate particular mono- and diterpenes. Under adequate photosynthetic conditions the chloroplastidic DOXP/MEP isoprenoid pathway essentially contributes, with its C5 isoprenoid precusors, to cytosolic sterol biosynthesis. The possible cross-talk between the two cellular isoprenoid pathways, the acetate/MVA and the DOXP/MEP pathways, that preferentially proceeds in a plastid-to-cytosol direction, is shortly discussed.     

The effect of class a scavenger receptor deficiency in bone

Class A scavenger receptor (SR-A) is predominantly expressed by macrophages, and because osteoclasts are of monocyte/macrophage lineage, SR-A is of potential interest in osteoclast biology. In addition to modified low density lipoprotein uptake, SR-A is also important in cell attachment and signaling. In this study we evaluated the effect of SR-A deletion on bone. Knock-out animals have 40% greater body weight than wild type. Body composition analyses demonstrated that total lean and fat body mass were greater in knock-out animals, but there was no significant difference in percent fat and lean body mass. Bone mineral density and content were significantly greater in knock-out compared with wild type animals. Micro-computed tomography analyses confirmed that total volume, bone volume as well as trabecular number, thickness, and connectivity were significantly greater in knock-out mice. As expected, trabecular separation was greater in wild type mice. The phenotype appears to be explained by 60% fewer osteoclasts in females and 35% fewer in males compared to wild type mice with a paradoxical increase in nuclei/osteoclast in knock-out animals. Furthermore, there were no differences in adipocyte number and osteoblast number or activity. The addition of the soluble extracellular domain of SR-A to RAW264.7 cells stimulated a concentration-dependent increase in osteoclast differentiation that was receptor activator of nuclear factor-kappaB ligand (RANKL)-dependent. Soluble SR-A had no effect on cell proliferation in the presence of RANKL but stimulated a 40% increase in numbers in the absence of RANKL. We conclude that SR-A plays a role in normal osteoclast differentiation, suggesting a novel role for this receptor in bone biology.     

Antibacterial functionalization of an experimental self-etching primer by inorganic agents: microbiological and biocompatibility evaluations

Antibacterial activities have been demonstrated on oral bacteria with inorganic antibacterial agents (ABAs) after their incorporations into an experimental self-etching primer (ESP) before curing. This study was to assess their biocompatibility and antibacterial activity after curing. Six ABAs were incorporated respectively into ESP for treating specimens. After curing, their bactericidal activities on Streptococcus mutans and influences to the early bacterial colonization were assessed by direct contact and viable count. Systemic toxicity in rats after short-term oral exposure and direct contact cytotoxicity with NIH3T3 fibroblasts were tested. Incorporation of ZnOw AT-83, Longbei antibiotic, Antim-AMS2 or IONPURE-H significantly enhanced the antibacterial effect of ESP after curing, even after 1 month aging. Specimens treated by ESP with ZnOw AT-83, Longbei antibiotic or Antim-AMS2 showed slightly less bacterial adhesion than control. Animal experiments revealed neither toxic signs nor significant differences in body weight gain between control and other groups. Cell vitality or proliferation rates were ranged from 76% to 100% with respect to controls. Basic magnesium hypochlorite, ZnOw AT-83 and ZnOw AT-88 were less toxic. Toxicity only observed in areas beneath the specimens and/or in the direct vicinity of the specimen edge. From microbiological and biocompatibility aspects, the tested ABAs can be effectively incorporated in ESP to provide antibacterial activity against S. mutans. ZnOw AT-83 was the most promising one.     

Selection of vimentin-specific antibodies from the HuCAL phage display library by subtractive panning on formalin-fixed, paraffin-embedded tissue

We describe the direct isolation of specific antibodies on formalin-fixed, paraffin-embedded (FFPE) tissue. The technique involves subtractive selection of a large and highly diverse combinatorial human antibody phage library (HuCAL) on lymphocyte FFPE tissue sections. Tissue sections from normal human tonsil tissue were used to deplete the library of binders to most housekeeping proteins. Mantle-cell lymphoma tissue was used for positive selection and enrichment of mantle cell or tumor-specific antibody phage. We established a high-throughput immunohistochemical method for screening of antibody clones selected from FFPE tissue. One recombinant antibody showed specific staining for interfollicular and mantle cells in FFPE tissue. Immunoprecipitation with this antibody and subsequent mass spectrometric analysis revealed specificity for vimentin.