Induction of CD69 expression and Th1 cytokines release from human peripheral blood lymphocytes after in vitro stimulation with Alloiococcus otitidis and three middle ear pathogens

Alloiococcus otitidis is a recently discovered pathogen of otitis media. However, only a limited number of studies are available about the pathogenic and immunological role of A. otitidis. The aim of this study was to investigate the activation and the cytokine production of human peripheral blood lymphocytes at the early immune response after stimulation with A. otitidis. After stimulation of whole human peripheral blood lymphocytes for 18 h with whole killed A. otitidis or the three major middle ear pathogens (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis), the expression of CD69 and the production of cytokines were analyzed. The expression of CD69 on T cells and B cells was dose-dependently enhanced after stimulation with A. otitidis. The release of interleukin (IL)-12 was induced after stimulation with A. otitidis, whereas the release of IL-4 was not induced after stimulation with A. otitidis. In addition, the release of interferon (IFN)-gamma was induced after stimulation with A. otitidis. Although the release of IFN-gamma started within 18 h after stimulation with A. otitidis, intracellular production of IFN-gamma was not observed in either CD4+ T cells or CD8+ T cells within 18 h upon stimulation. The patterns of CD69 expression and T helper-type 1 (Th1)-promoting cytokines production were similarly shown when human peripheral blood lymphocytes were stimulated with the other three major pathogens. Our results suggest that A. otitidis has sufficient immunogenic potential to modulate a host immune response, like the other three major middle ear pathogens, and also suggest that the immunogenicity of A. otitidis is very similar, at the early immune response, to that of the three major middle ear pathogens.     

The FtsH protease heterocomplex in Arabidopsis: dispensability of type-B protease activity for proper chloroplast development

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.     

Mechanical stretch stimulates integrin αVβ3-mediated collagen expression in human anterior cruciate ligament cells

Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Here, we focused on the stretch-related responses of different cells derived from the ligament-to-bone interface and midsubstance regions of human anterior cruciate ligaments. Chondroblastic interface cells easily lost their potential to produce collagen genes in non-stretched conditions, rather than fibroblastic midsubstance cells. Uni-axial mechanical stretches increased the type I collagen gene expression of interface and midsubstance cells up to 14- and 6-fold levels of each non-stretched control, respectively. Mechanical stretches also activated the stress fiber formation by shifting the distribution of integrin αVβ3 to the peripheral edges in both interface and midsubstance cells. In addition, integrin αVβ3 colocalized with phosphorylated focal adhesion kinase in stretched cells. Functional blocking analyses using anti-integrin antibodies revealed that the stretch-activated collagen gene expressions on fibronectin were dependent on integrin αVβ3-mediated cellular adhesions in the interface and midsubstance cells. These findings suggest that the integrin αVβ3-mediated stretch signal transduction might have a key role to stimulate collagen gene expression in human anterior cruciate ligament, especially in the ligament-to-bone interface.     

Streptococcus panodentis sp. nov. from the oral cavities of chimpanzees

Three strains TKU9, TKU49 and TKU50^T, were isolated from the oral cavities of chimpanzees (Pan troglodytes). The isolates were all gram‐positive, facultative anaerobic cocci that lacked catalase activity. Analysis of partial 16S rRNA gene sequences showed that the most closely related species was Streptococcus infantis (96.7%). The next most closely related species to the isolates were S. rubneri, S. mitis, S. peroris and S. australis (96.6 to 96.4%). Based on the rpoB and gyrB gene sequences, TKU50^T was clustered with other member of the mitis group. Enzyme activity and sugar fermentation patterns differentiated this novel bacterium from other members of the mitis group streptococci. The DNA G + C content of strain TKU50^T was 46.7 mol%, which is the highest reported value for members of the mitis group (40–46 mol%). On the basis of the phenotypic characterization, partial 16S rRNA gene and sequences data for two housekeeping gene (gyrB and rpoB), we propose a novel taxa, S. panodentis for TKU 50^T (type strain = CM 30579^T = DSM 29921^T), for these newly described isolates.     

Inhibitory Effect of Ascorbic Acid on the Proliferation and Invasion of Hepatoma Cells in Culture

Effect of ascorbic acid (AsA) on the proliferation and invasion of rat ascites hepatoma AH109A cells was investigated by measuring [³H]thymidine incorporation into acid-insoluble fraction of the cells and by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells, respectively. AsA suppressed the invasion of AH109A cells in a dose-dependent manner at concentrations of 62.5–500 μM, while it inhibited the proliferation of the cells at higher concentrations of 250 and 500 μM. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. AsA suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with AsA, HX and XO or with AsA and hydrogen peroxide. Furthermore, AsA reduced the intracellular peroxide levels in AH109A cells. These results suggest that the antioxidative property of AsA may be involved in its anti-invasive action on hepatoma cells.     

Molecular Conformation of 2′-Deoxy-2′-methylidene-cytidine: A Potent Antineoplastic Nucleoside

Abstract

2′-Deoxy-2′-methylidenecytidine (DMDC), a potent inhibitor of the growth of tumor cells, was crystallized with two different forms. One is dihydrated (DMDC·2H₂O) and the other is its hydrochloride salt (DMDC·HCLl). Both crystal and molecular structures have been determined by the X-ray diffraction method. In both forms the glycosidic and sugar conformations are anti and C(4′)-exo, respectively, whereas the conformation about the exocyclic bond is trans for DMDC·2H₂O and gauche ⁺ for DMDC·HCl. Proton nuclear magnetic resonance data of DMDC indicate a preference for the anti C(4′)-exo conformation found in the solid state. These molecular conformations were compared with the related pyrimidine nucleosides. When the cytosine bases are brought into coincidence, DMDC displays the exocyclic C(4′)-C(5′) bond located on the very close position to those of pyrimidine nucleosides with typical overall conformations. On the other hand, the hydroxyl O(3′)-H groups are separated by ca. 3 Å in the cases of DMDC and other pyrimidine nucleosides which have the C(2′)-endo sugar conformation. This result may be useful for the implication about the mechanism of the biological activity of DMDC.     

Brain Endogenous Estrogen Levels Determine Responses to Estrogen Replacement Therapy via Regulation of BACE1 and NEP in Female Alzheimer’s Transgenic Mice

Estrogens have been found to improve memory and reduce risk of dementia, although conflicting results such as failure of estrogen replacement therapy for treatment of Alzheimer's disease (AD) also has been reported. Only recently, our published human brain studies showed a depletion of brain estrogen in women with AD, while other studies have demonstrated cognitive impairment believed to be caused by inhibition of endogenous estrogen synthesis in females. To investigate whether the shortage of brain estrogen alters the sensitivity of response to estrogen replacement therapy, we have used genetic and surgical animal models to examine the response of estrogen treatment in AD neuropathology. Our studies have shown that early treatment with 17β-estradiol (E2) or genistein could reduce brain amyloid levels by increasing Aβ clearance in both APP23 mice with genetic deficiency of aromatase (APP/Ar^+/?), in which the brains contain nondetectable levels of estrogen, and in APP23 mice with an ovariectomy (APP/OVX), in which the brains still contain certain levels of estrogen. However, only APP/Ar^+/? mice showed a great reduction in brain amyloid plaque formation after E2 or genistein treatment along with downregulation of β-secretase (BACE1) mRNA and protein expression. Our results suggest that early and long-term usage of E2 and/or genistein may prevent AD pathologies in a dependent manner on endogenous brain estrogen levels in aged females.     

Purification and Properties of Dimethylglycine Oxidase from Cylindrocarpon didymum M–1

Dimethylglycine oxidase was purified to homogeneity from the cell extract of Cylindrocarpon didymum M–1, aerobically grown in medium containing betaine as the carbon source. The molecular weight of the enzyme was estimated to be 170,000 by the gel filtration method and 180,000 by the sedimentation velocity method. The enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 277, 345 and 450 nm. The enzyme consisted of two identical subunits with a molecular weight of 82,000, and contained two mol of FAD per mol of enzyme. The flavin was shown to be covalently bound to the protein. The enzyme was inactivated by Ag⁺, Hg²⁺, Zn²⁺ and iodoacetate. The enzyme oxidized dimethylglycine but was inert toward choline, betaine, sarcosine and alkylamines. Km and Vmax values for dimethylglycine were 9.1 mm and 1.22 μmol/min/mg, respectively. The enzyme catalyzed the following reaction: Dimethylglycine+O₂+H₂O → sarcosine+formaldehyde+H₂O₂.     

Large-scale profiling of brown rice ionome in an ethyl methanesulphonate-mutagenized hitomebore population and identification of high- and low-cadmium lines

Background and aim

Recycled sources of phosphorus (P), such as struvite extracted from wastewater, have potential to substitute for more soluble manufactured fertilisers and help reduce the long-term threat to food security from dwindling finite reserves of phosphate rock (PR). This study aimed to determine whether struvite could be a component of a sustainable P fertiliser management strategy for arable crops.

Methods

A combination of laboratory experiments, pot trials and mathematical modelling of the root system examined the P release properties of commercial fertiliser-grade struvite and patterns of P uptake from a low-P sandy soil by two different crop types, in comparison to more soluble inorganic P fertilisers (di-ammonium phosphate (DAP) and triple super phosphate (TSP)).

Results

Struvite had greatly enhanced solubility in the presence of organic acid anions; buckwheat, which exudes a high level of organic acids, was more effective at mobilising struvite P than the low level exuder, spring wheat. Struvite granules placed with the seed did not provide the same rate of P supply as placed DAP granules for early growth of spring wheat, but gave equivalent rates of P uptake, yield and apparent fertiliser recovery at harvest, even though only 26 % of struvite granules completely dissolved. Fertiliser mixes containing struvite and DAP applied to spring wheat have potential to provide both optimal early and late season P uptake and improve overall P use efficiency.

Conclusions

We conclude that the potential resource savings and potential efficiency benefits of utilising a recycled slow release fertiliser like struvite offers a more sustainable alternative to only using conventional, high solubility, PR-based fertilisers.

     

Freeze-dried platelets promote hepatocyte proliferation in mice

In recent years, platelets are reported to promote liver, as well as bone regeneration and dermal wound healing. Platelets are required not only for thrombocytopenia treating but also for regenerative therapy. Platelets cannot be stored beyond three days, therefore, shortage of fresh platelets sometimes occurs. To preserve platelets for a long duration without degrading growth factors, a freeze-dried technique is required. We report here that platelets can be preserved by freeze-drying, using a programmed freezing method to avoid intracellular ice crystal formation. Freeze-dried platelets kept their morphological countenance and response with the agonist of thrombin was well maintained. Freeze-dried platelets stored adenine nucleotides, PDGF, and IGF-1 the same as those of fresh platelets. Freeze dried platelets also preserved their proliferative effect on hepatocytes identical to that of fresh platelets. These results of our study suggest that freeze dried platelets will obviate the storage problem of fresh platelets.