<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Regulation of IL-11 expression in intestinal myofibroblasts: role of c-Jun AP-1- and MAPK-dependent pathways

IL-11 inhibits the activation of NF-kappaB and induces the Th2 polarization of CD4+ T cells. The clinical utility of IL-11 is being investigated in Crohn's disease. However, physiological secretion of IL-11 in the intestine remains unclear. In this study, we investigated IL-11 secretion in human intestinal subepithelial myofibroblasts (SEMFs). Intestinal SEMFs were isolated from the human colonic mucosa. IL-11 secretion and mRNA expression were determined by ELISA and Northern blot analysis. The activating protein (AP)-1-DNA binding activity was evaluated by EMSA. IL-11 secretion was induced by IL-1beta and transforming growth factor (TGF)-beta1. These were also observed at the mRNA level. The EMSAs demonstrated that both IL-1beta and TGF-beta1 induced AP-1 activation within 2 h after stimulation, and a blockade of AP-1 activation by the recombinant adenovirus containing a dominant negative c-Jun markedly reduced the IL-1beta- and TGF-beta1-induced IL-11 mRNA expression. IL-1beta and TGF-beta1 induced an activation of ERK p42/44 and p38 MAP kinases, and the MAP kinase inhibitors (SB-202190, PD-98059, and U-0216) significantly reduced the IL-1beta- and TGF-beta1-induced IL-11 secretion. The upregulation of IL-11 mRNA by IL-1beta- and TGF-beta1 was also mediated by a p38 MAP kinase-mediated mRNA stabilization. The combination of IL-1beta and TGF-beta1 additively enhanced IL-11 secretion. Intestinal SEMFs secreted IL-11 in response to IL-1beta- and TGF-beta1. Mucosal IL-11 secretion might be important as an anti-inflammatory response in the pathogenesis of intestinal inflammation.     

ICRF-193, an Inhibitor of Topoisomerase II, Demonstrates That DNA Replication in Sperm Nuclei Reconstituted in Xenopus Egg Extracts Does Not Require Chromatin Decondensation

A bis(2,6-dioxopiperazine) derivative, ICRF-193, is a specific inhibitor of topoisomerase II without clearable complex-stabilizing activity. In Xenopus egg extract containing ICRF-193, demembranated sperm head chromatins were inhibited from decondensation. However, nuclear envelope-lamina assembled on the inhibited chromatins. The nuclear envelope-lamina continued to expand even after loss of contact with the chromatin surface. On the other hand, semiconservative DNA replication was initiated as soon as the lamina was assembled onto the surface of condensed chromatin, though the initiation was retarded and its extent was reduced, compared with that in noninhibited chromatins. Thus, it is concluded that topoisomerase II activity is not required for the formation of active DNA replication clusters and the extension of nuclear envelope-lamina on the chromatin, while the nuclear envelope-mediated decondensation of sperm chromatins is dependent on topoisomerase II activity.     

Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

Cytomegaloviruses manipulate the host chemokine/receptor axis by altering cellular chemokine expression and by encoding multiple chemokines and chemokine receptors. Similar to human cytomegalovirus (HCMV), rat cytomegalovirus (RCMV) encodes multiple CC chemokine-analogous proteins, including r129 (HCMV UL128 homologue) and r131 (HCMV UL130 and MCMV m129/130 homologues). Although these proteins play a role in CMV entry, their function as chemotactic cytokines remains unknown. In the current study, we examined the role of the RCMV chemokine r129 in promoting cellular migration and in accelerating transplant vascular sclerosis (TVS) in our rat heart transplant model. We determined that r129 protein is released into culture supernatants of infected cells and is expressed with late viral gene kinetics during RCMV infection and highly expressed in heart and salivary glands during in vivo rat infections. Using the recombinant r129 protein, we demonstrated that r129 induces migration of lymphocytes isolated from rat peripheral blood, spleen, and bone marrow and from a rat macrophage cell line. Using antibody-mediated cell sorting of rat splenocytes, we demonstrated that r129 induces migration of naïve/central memory CD4⁺ T cells. Through ligand-binding assays, we determined that r129 binds rat CC chemokine receptors CCR3, CCR4, CCR5, and CCR7. In addition, mutational analyses identified functional domains of r129 resulting in recombinant proteins that fail to induce migration (r129-ΔNT and -C31A) or alter the chemotactic ability of the chemokine (r129-F43A). Two of the mutant proteins (r129-C31A and -ΔNT) also act as dominant negatives by inhibiting migration induced by wild-type r129. Furthermore, infection of rat heart transplant recipients with RCMV containing the r129-ΔNT mutation prevented CMV-induced acceleration of TVS. Together our findings indicate that RCMV r129 is highly chemotactic, which has important implications during RCMV infection and reactivation and acceleration of TVS.     

The short-term effects of various oral care methods in dependent elderly: comparison between toothbrushing, tongue cleaning with sponge brush and wiping on oral mucous membrane by chlorhexidine

doi: 10.1111/j.1741‐2358.2011.00577.x The short‐term effects of various oral care methods in dependent elderly: comparison between toothbrushing, tongue cleaning with sponge brush and wiping on oral mucous membrane by chlorhexidine Objectives: To explore the short‐term effects from toothbrushing, tongue cleaning with sponge brush and wiping on oral mucous membrane by chlorhexidine. Background: Numerous reports have been seen in recent years proving the effectiveness of mouth cleaning with a toothbrush for the prevention of respiratory infections among the dependent elderly. However, the short‐term effects from each oral care method have not yet been clarified. Hence, an investigation was conducted by having each subject independently perform various oral care methods for five consecutive days. Materials and Methods: The subjects consisted of 12 assistance‐dependent elderly who have difficulties with tooth brushing by themselves, have 10 or more residual teeth and are not yet using plate dentures. After the pre‐intervention examination, each of the following oral care methods were performed on the same subject on an approximately three week basis: 1) Tooth brushing 2)Tongue cleaning with sponge brush 3)Wiping on oral mucous with sponge brush by chlorhexidine. Each method was performed independently, once a day for 5 consecutive days and the subjects were reexamined on the sixth day for comparative verification. Results: Consequently, toothbrushing decreased the plaque index and gingival index significantly and an improvement of oral malodour was also acknowledged (p < 0.01). Tongue cleaning with a sponge brush decreased the tongue coat score significantly (p < 0.05) and oral malodour was also improved (p < 0.01). Wiping on oral mucous with a sponge brush soaked in chlorhexidine significantly decreased opportunistic infections in the pharynx region (p < 0.05). Conclusions: It was suggested that the use of not only a toothbrush but also chlorhexidine gluconate may be indicated for dependent elderly people in whom pathogens of opportunistic infection are detected.     

Interleukin-32 Expression in the Pancreas

Interleukin (IL)-32 is a recently described proinflammatory cytokine characterized by the induction of nuclear factor (NF)-κB activation. We studied IL-32 expression in human pancreatic tissue and pancreatic cancer cell lines. Tissue samples were obtained surgically. IL-32 expression was evaluated by standard immunohistochemical procedures. IL-32 mRNA expression was analyzed by Northern blotting and real time PCR analyses. IL-32 was weakly immunoexpressed by pancreatic duct cells. In the inflamed lesions of chronic pancreas, the ductal expression of IL-32 was markedly increased. A strong expression of IL-32α was detected in the pancreatic cancer cells. In pancreatic cancer cell lines (PANC-1, MIA PaCa-2, and BxPC-3 cells), the expression of IL-32 mRNA and protein was enhanced by IL-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. An inhibitor of phosphatidylinositol 3-kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) significantly suppressed the IL-1β-, IFN-γ- and TNF-α-induced IL-32 mRNA expression. The blockade of NF-κB and activated protein-1 activation markedly suppressed the IL-1β-, IFN-γ-, and/or TNF-α-induced IL-32 mRNA expression. Furthermore, IL-32-specific small interfering RNA significantly decreased the uptake of [³H]thymidine and increased the annexin V-positive population (apoptotic cells) in PANC-1 cells. IL-32 knockdown also suppressed the mRNA expression of antiapoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1). Pancreatic duct cells are the local source of IL-32, and IL-32 may play an important role in inflammatory responses and pancreatic cancer growth.     

Oxidative stress increases MICA and MICB gene expression in the human colon carcinoma cell line (CaCo-2)

The human major histocompatibility complex class I chain-related A gene (MICA) and the MICB gene are newly identified members of the major histocompatibility complex class I chain-related gene family. We demonstrate here that oxidative stress, induced by H(2)O(2), promoted MICA (2.2-fold) and MICB (3.8-fold) gene expression using the human colon carcinoma cell line (CaCo-2) and semi-quantitative RT-PCR.