Thermal conversion of alkaline lignin and its structured derivatives to porous carbonized materials

Alkaline lignin was thermally converted to microporous carbon in ca. 50% yield by heating up from room temperature to 900 °C without activation process under flowing of an argon gas. The carbonized material prepared by heating up conditions of 1 °C min⁻¹ showed 530 m²/g of the Brunauer-Emmett-Teller (BET) specific surface area, which increased to 740 m²/g after washing with water. Furthermore, alkaline lignin derivatives were structured as micron scale particles by micelle formation and polymer gelation techniques. Carbonization of the structured lignins could afford high porous materials having BET surface areas above 1000 m²/g without surface activation processes.     

Clonal Rett Syndrome cell lines to test compounds for activation of wild-type MeCP2 expression

Rett Syndrome is an X-linked progressive neurological disorder caused by inactivation of one allele of the MECP2 gene. There are no curative treatments, and activation of wild-type MECP2 expression is one strategy for stabilizing or reversing the disease. We isolated fibroblast clones that express exclusively either the wild-type or a 32-bp-deletion mutant form of MECP2. We developed a sensitive assay for measuring wild-type MECP2 mRNA levels and tested small molecule epigenetic activators for their ability to activate gene expression. Although our pilot screen did not identify activators of MECP2 expression, it established the value of using clonal cells and defined challenges that must be overcome.     

Calmodulin-dependent activation of MAP kinase for ROS homeostasis in Arabidopsis

Rapid recognition and signal transduction of mechanical wounding through various signaling molecules, including calcium (Ca2+), protein phosphorylation, and reactive oxygen species (ROS), are necessary early events leading to stress resistance in plants. Here we report that an Arabidopsis mitogen-activated protein kinase 8 (MPK8) connects protein phosphorylation, Ca2+, and ROS in the wound-signaling pathway. MPK8 is activated through mechanical wounding, and this activation requires direct binding of calmodulins (CaMs) in a Ca2+-dependent manner. MPK8 is also phosphorylated and activated by a MAPKK MKK3 in the prototypic kinase cascade, and full activation of MPK8 needs both CaMs and MKK3 in planta. The MPK8 pathway negatively regulates ROS accumulation through controlling expression of the Rboh D gene. These findings suggest that two major activation modes in eukaryotes, Ca2+/CaMs and the MAP kinase phosphorylation cascade, converge at MPK8 to monitor or maintain an essential part of ROS homeostasis.     

Functional morphology of the metapleural gland in workers of the ant Crematogaster inflata (Hymenoptera,Formicidae)

Abstract. Workers of Crematogaster inflata possess the largest metapleural glands (relative to body size) known among ants, with reservoirs extending anteriorly up to the junction between the pro‐ and the mesothorax, and with over 1400 secretory cells on both sides together. This large secretory capacity is related to the gland's defensive function, which, in members of this species, is directed against larger arthropod and vertebrate enemies, and apparently not against microorganisms, in contrast to other ants, where the gland produces antibiotics. The gland is not equipped with any direct musculature. Secretion release is probably caused by contraction of the oblique longitudinal thorax muscles or by passive expulsion caused by external pressure.     

Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.     

Inhibition of neuronal nitric-oxide synthase by phosphorylation at Threonine1296 in NG108-15 neuronal cells

We demonstrate that neuronal nitric-oxide synthase (nNOS) is directly inhibited through the phosphorylation of Thr(1296) in NG108-15 neuronal cells. Treatment of NG108-15 cells expressing nNOS with calyculin A, an inhibitor of protein phosphatase 1 and 2A, revealed a dose-dependent inhibition of nNOS enzyme activity with concomitant phosphorylation of Thr(1296) residue. Cells expressing a phosphorylation-deficient mutant in which Thr(1296) was changed to Ala proved resistant to phosphorylation and suppression of NOS activity. Mimicking phosphorylation mutant of nNOS in which Thr(1296) is changed to Asp showed a significant decrease in nNOS enzyme activity, being competitive with NADPH, relative to the wild-type enzyme. These data suggest that phosphorylation of nNOS at Thr(1296) may involve the attenuation of nitric oxide production in neuronal cells through the decrease of NADPH-binding to the enzyme.     

Eotaxin-3/CC chemokine ligand 26 is a functional ligand for CX3CR1

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1.     

Novel plastocyanin containing phenylalanine-83 from the gymnosperm Ginkgo biloba

Plastocyanin was purified from the gymnosperm Ginkgo biloba L., and its complete amino acid sequence was determined. The protein was shown to contain Phe-83 instead of Tyr-83 conserved in other land plant plastocyanins. This is the first report of the characterization and complete amino acid sequence of a gymnosperm plastocyanin.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

Involvement of IL-10 and Bcl-2 in resistance against an asbestos-induced apoptosis of T cells

To analyze the possibility that immunological alteration in asbestos-related diseases (ARDs) such as asbestosis (ASB) and malignant mesothelioma (MM) may affect the progression of cancers, a human adult T cell leukemia virus-immortalized T cell line (MT-2Org) was continuously exposed to 10 μg/ml of chrysotile-B (CB), an asbestos. After at least 8 months of exposure, the rate of apoptosis in the cells became very low and the resultant subline was designated MT-2Rst. The MT-2Rst cells were characterized by (i) enhanced expression of bcl-2, with regain of apoptosis-sensitivity by reduction of bcl-2 by siRNA, (ii) excess IL-10 secretion and expression, and (iii) activation of STAT3 that was inhibited by PP2, a specific inhibitor of Src family kinases. These results suggested that the contact between cells and asbestos may affect the human immune system and trigger a cascade of biological events such as activation of Src family kinases, enhancement of IL-10 expression, STAT3 activation and Bcl-2 overexpression. This speculation was partially confirmed by the detection of elevated bcl-2 expression levels in CD4 + peripheral blood T cells from patients with MM compared with those from patients with ASB or healthy donors. Further studies will be required to verify the role of T cells with enhanced bcl-2 expression in tumor progression induced by asbestos exposure.