Cellular basis of the role of diesel exhaust particles in inducing Th2-dominant response

There is growing evidence that diesel exhaust particles (DEP) can induce allergic diseases with increased IgE production and preferential activation of Th2 cells. To clarify the cellular basis of the role of DEP in the induction of Th2-dominant responses, we examined the effects of DEP on the cytokine production by T cells stimulated with anti-CD3/CD28 Ab and on that by monocyte-derived dendritic cells (MoDCs) stimulated with CD40L and/or IFN-gamma. We examined IFN-gamma, IL-4, IL-5, IL-8, and IL-10 produced by T cells and TNF-alpha, IL-1beta, IL-10, and IL-12 produced by MoDCs using real-time PCR analysis or by ELISA. To highlight the effects of DEP, we compared the effects of DEP with those of dexamethasone (DEX) and cyclosporin A (CyA). DEP significantly suppressed IFN-gamma mRNA expression and protein production, while it did not affect IL-4 or IL-5 mRNA expression or protein production. The suppressive effect on IFN-gamma mRNA expression was more potent than that of DEX and comparable at 30 mug/ml with 10(-7) M CyA. The suppressive effect on IFN-gamma production was also more potent than that of either DEX or CyA. DEP suppressed IL-12p40 and IL-12p35 mRNA expression and IL-12p40 and IL-12p70 production by MoDCs, while it augmented IL-1beta mRNA expression. Finally, by using a thiol antioxidant, N-acetyl cysteine, we found that the suppression of IFN-gamma production by DEP-treated T cells was mediated by oxidative stress. These data revealed a unique characteristic of DEP, namely that they induce a Th2 cytokine milieu in both T cells and dendritic cells.     

Evolutional conservation of molecular structure and antiviral function of a viral RNA receptor, LGP2, in Japanese flounder, Paralichthys olivaceus

LGP2 is an important intracellular receptor that recognizes viral RNAs in innate immunity. To understand the mechanism of viral RNA recognition, we cloned an LGP2 cDNA and gene in Japanese flounder (Paralichthys olivaceus). Viral hemorrhagic septicemia virus-induced expressions of LGP2 mRNA were evaluated in vivo and in vitro by quantitative real-time PCR (Q-PCR) using primers based on the clone sequences. The expression of LGP2 mRNA in the kidney dramatically increased at 3 d postinfection. The expression of LGP2 mRNA also increased in the head kidney leukocytes stimulated with artificial dsRNA (polyinosin-polycytidylic acid) in vitro. To evaluate the antiviral activity of the flounder LGP2, three expression constructs containing pcDNA4-LGP2 (full-length), pcDNA4-LGP2ΔRD (regulatory domain deleted), and pcDNA4-Empty (as a negative control) were transfected into the hirame (flounder) natural embryo (hirame natural embryo) cell line. Forty-eight hours after transfection, the transfected cells were infected with ssRNA viruses, viral hemorrhagic septicemia virus, or hirame rhabdovirus. The cytopathic effects of the viruses were delayed by the overexpression of Japanese flounder LGP2. The Q-PCR demonstrated that mRNA expression levels of type I IFN and IFN-inducible genes (Mx and ISG15) in the hirame natural embryo cells overexpressing LGP2 were increased by polyinosin-polycytidylic acid and viral infections. These results suggest that Japanese flounder LGP2 plays an important role in the recognition of both viral ssRNA and dsRNA to induce the antiviral activity by the production of IFN-stimulated proteins.     

A novel Delta9 acyl-lipid desaturase, DesC2, from cyanobacteria acts on fatty acids esterified to the sn-2 position of glycerolipids

Acyl-lipid desaturases are enzymes that convert a C-C single bond into a C=C double bond in fatty acids that are esterified to membrane-bound glycerolipids. Four types of acyl-lipid desaturase, namely DesA, DesB, DesC, and DesD, acting at the Delta12, Delta15, Delta9, and Delta6 positions of fatty acids respectively, have been characterized in cyanobacteria. These enzymes are specific for fatty acids bound to the sn-1 position of glycerolipids. In the present study, we have cloned two putative genes for a Delta9 desaturase, designated desC1 and desC2, from Nostoc species. The desC1 gene is highly similar to the desC gene that encodes a Delta9 desaturase that acts on C18 fatty acids at the sn-1 position. Homologues of desC2 are found in genomes of cyanobacterial species in which Delta9-desaturated fatty acids are esterified to the sn-2 position. Heterologous expression of the desC2 gene in Synechocystis sp. PCC 6803, in which a saturated fatty acid is found at the sn-2 position, revealed that DesC2 could desaturate this fatty acid at the sn-2 position. These results suggest that the desC2 gene is a novel gene for a Delta9 acyl-lipid desaturase that acts on fatty acids esterified to the sn-2 position of glycerolipids.     

Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33°C. A temperature shift to 37°C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.M. Matsuo and K. Koizumi contributed equally to this work. This study was supported in part by Grants-in-Aid for the 21st Century COE Program and for CLUSTER (Cooperative Link of Unique Science and Technology for Economy Revitalization) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.     

Reaction on D-glucal by an inverting phosphorylase to synthesize derivatives of 2-deoxy-beta-D-arabino-hexopyranosyl-(1-->4)-D-glucose (2II-deoxycellobiose)

Four derivatives of 2(II)-deoxycellobiose were synthesized from d-glucal and acceptor sugars (d-glucose, d-xylose, d-mannose, and 2-deoxy-d-arabino-hexose) using a cellobiose phosphorylase from Cellvibrio gilvus. The enzyme was found to be an effective catalyst to synthesize the beta-(1-->4) linkage of 2-deoxy-d-arabino-hexopyranoside. The acceptor specificity for the d-glucal reaction was identical to that for the alpha-d-glucose 1-phosphate reaction, but the activity of d-glucal was approximately 500 times less than that of alpha-d-glucose 1-phosphate, using 10mM substrates.     

Cyclophosphamide enhances TNF-alpha-induced apoptotic cell death in murine vascular endothelial cell

Cyclophosphamide (CPA) is one of the therapeutic agents for systemic inflammatory disorders. In murine dermal endothelial cells (F-2), 4-hydroxycyclophosphamide (4-HC), which is active metabolite of CPA, enhanced TNF-alpha-induced DNA fragmentation. In addition, 4-HC was shown to elevate TNF-alpha-induced caspase-3 activation. Caspase-8 activation was identified by the treatment of TNF-alpha, whereas 4-HC was no effect. In contrast, only when treated with 4-HC, caspase-9 activation and the increase in the intracellular expression of Bax were detected. These results suggest that CPA may sensitize endothelial cells to TNF-alpha-induced apoptosis through a mitochondria-dependent pathway and clinically may contribute to the limitation of inflammatory process.     

Colonization Dynamics of Altered Schaedler Flora Is Influenced by Gender, Aging, and Helicobacter hepaticus Infection in the Intestines of Swiss Webster Mice

The distribution and colonization levels of the altered Schaedler flora (ASF) in their natural hosts are poorly understood. Intestinal colonization levels of the eight ASF strains in outbred Swiss Webster mice with or without Helicobacter hepaticus infection were characterized by real-time quantitative PCR. All ASF strains were detected in the cecum and colon, but some strains displayed significant variation in colonization levels with host age, gender, and H. hepaticus infection status.     

Electric and magnetic fields in cryopreservation: A response

Our recent studies showed that a programmed freezer with a magnetic field (CAS freezer) is helpful in the survival of periodontal ligament (PDL) cells after cryopreservation. The theory is that a magnetic field can prevent the cluster from growing by causing it to vibrate. In this letter, we commented in detail on the influence of a magnetic field during cryopreservation.     

Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: a BAC-based domino method applied to DNA with a high GC content

Bacillus subtilis is the only bacterium-based host able to clone giant DNA above 1000 kbp. DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. To expand the target DNA range to higher GC content, we tried to clone a pTT27 megaplasmid (257 kbp, 69% of G+C) from Thermus thermophilus. To facilitate the reconstruction process, we subcloned pTT27 in a bacterial artificial chromosome (BAC) vector of Escherichia coli. Owing to the ability of BAC to carry around 100 kbp DNA, only 4 clones were needed to cover the pTT27 and conduct step-by-step assembly in the B. subtilis genome. The full length of 257 kbp was reconstructed through 3 intermediary lengths (108, 153, and 226 kbp), despite an unexpected difficulty in the maintenance of DNA >200 kbp. Retrieval of these four pTT27 segments from the B. subtilis genome by genetic transfer to a plasmid pLS20 was attempted. A stable plasmid clone was obtained only for the 108 and 153 kbp intermediates. The B. subtilis genome was demonstrated to accommodate large DNA with a high GC content, but may be restricted to less than 200 kbp by unidentified mechanisms.