Toll-like receptor 2 is expressed on the intestinal M cells in swine

The Toll-like receptor (TLR) 2 binds a wide variety of microbial cell wall components. In this study, we investigated the expression pattern of TLR2 in adult swine gut-associated lymphoid tissues using real-time quantitative PCR, Western blotting, immunohistochemistry, and flow cytometric analysis. The mRNA for TLR2 was preferentially expressed in the mesenteric lymph nodes (MLNs) and Peyer's patches (Pps) of adult swine. Expression in these two tissues was approximately 15- and 9-fold higher than that of spleen, respectively. Western blotting further confirmed that the TLR2 protein was highly expressed in the MLNs and Pps. Interestingly, TLR2-expressing cells were found not only in immune cells, such as T cells and B cells, but also in membranous (M) cells. In addition, double immunostaining for TLR2 and cytokeratin 18 revealed that TLR2 was strongly expressed not only in the cytoplasm but also in the apical membrane of the pocket-like M cells. These results indicate that TLR2 on the MLNs and Pps enable the host defense to respond to a variety of cell wall components. Furthermore, the potential function of TLR2 as a pattern recognition receptor and its cellular distribution suggest that TLR2 plays an important role in ligand-specific transcytosis and transport in M cells.     

Expression of P-cadherin distinct from that of E-cadherin in re-epithelialization in neonatal rat skin

Our previous study showed that an open wound made in neonatal rat skin was covered by migration of certain undifferentiated populations of keratinocytes as a multilayered cell sheet. In this study, the expression of the components of adherens junctions (AJ), E- and P-cadherins, and beta-catenin, was examined to understand the underlying mechanisms. Both E- and P-cadherins were downregulated in the basal layer at 6 h post-wounding (PW), indicating a reduction in the intercellular adhesiveness. The expression of P-cadherin but not E-cadherin was expanded to the suprabasal layers at the wound margin at 12 h PW. Moreover, the expression pattern of P-cadherin at sites of cell-cell contact was punctate rather than linear. By 24 h PW, cells accumulated beta-catenin in the cytoplasm in a suprabasal layer contacting the basal layer at the wound margin. Both the E- and P-cadherins showed a punctate AJ pattern at the confined suprabasal layer. Such differential expression of the E- and P-cadherins strongly suggests that these two classic cadherins play distinct roles in re-epithelialization. The changing of the E- and/or P-cadherin expression may participate in a delay of terminal differentiation of keratinocytes for cell supply toward a wound.     

The synthetic peptide derived from the NH2-terminal extracellular region of an orphan G protein-coupled receptor, GPR1, preferentially inhibits infection of X4 HIV-1

Several G protein-coupled receptors (GPCRs) serve as co-receptors for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. Here we report that a synthetic peptide derived from the NH2-terminal extracellular region of an orphan GPCR, GPR1 (GPR1ntP-(1-27); MEDLEETLFEEFENYSYDLDYYSLESC), inhibited infection of not only an HIV-1 variant that uses GPR1 as a co-receptor, but also X4, R5, and R5X4 viruses. Among these HIV-1 strains tested, viruses that can utilize CXCR4 as their co-receptors were preferentially inhibited. Inhibition of early steps in X4 virus replication was also detected in the primary human peripheral blood lymphocytes. GPR1ntP-(1-27) directly interacted with recombinant X4 envelope glycoprotein (rgp120). This interaction was neither inhibited nor enhanced by the soluble CD4 (sCD4) but inhibited by the anti-third variable (V3) loop-specific monoclonal antibody and heparin known to bind to the V3 loop. Although the conformational changes in gp120, including the V3 loop, have been reported to be required for its interaction with a co-receptor after binding of gp120 to CD4, it has also been reported that the V3 loop is already exposed on the surface of virions before interaction with CD4. We found that GPR1ntP-(1-27) blocked binding of virus to the cells, and this peptide equally bound to rgp120 in the presence or absence of sCD4. Because we detected the binding of GPR1ntP-(1-27) to the highly purified virions even in the absence of sCD4, GPR1ntP-(1-27) probably recognized the V3 loop exposed on the virions, and this interaction was responsible for the anti-HIV-1 activity of GPR1ntP-(1-27).     

Synergistic effect of interleukin-15 and interleukin-12 on antitumor activity in a murine malignant pleurisy model

 Interleukin(IL)-15, which uses IL-2 receptor (R) β and γ chains for signal transduction, shares many of the biological activities of IL-2. We examined the effects of exogenous IL-15 on protection in a murine malignant pleurisy model using BALB/c mice and syngeneic MethA fibrosarcoma (MethA). Intrapleural administration of IL-15 significantly prolonged the survival time of mice after an intrapleural inoculation of MethA, whereas the same dose of IL-2 did not. The in vivo antitumor effect of IL-15 was synergistically enhanced by additive administration of IL-12. Combination therapy of IL-15 and IL-12 protected mice from death from bloody pleural fluid. Such treatment induced marked increases in the number of CD3^-IL-2Rβ⁺ cells corresponding to natural killer (NK) cells and the production of interferon γ (IFNγ) by T cells in the thoracic exudate cells (TEC). Administration of anti-IFNγ mAb partly inhibited the protective effect of a combination of IL-15 and IL-12. A tumor-neutralizing (Winn) assay revealed that the antitumor activity of effector cells in the TEC was abrogated by treatment with anti-CD8 mAb or anti-asialoGM1 Ab plus complement. Thus, treatment with IL-15 in combination with IL-12 may enhance the activities of NK and CD8⁺ T cells in the TEC, providing strong antitumor activity against the malignant pleurisy. These results suggest that IL-15 together with IL-12 may have potential for the immunotherapy of some types of malignant pleurisy. Received: 13 July 1999 / Accepted: 3 December 1999     

Efficient light harvesting through carotenoids

We review the factors that control the efficiency of carotenoid-chlorophyll excitation transfer in photosynthetic light harvesting. For this we summarize first the recently developed theory that describes electronic couplings between carotenoids and chlorophylls and we outline in particular the influence of length of conjugated system and of symmetry breaking on the couplings. We focus hereby on the structurally solved lycopene-BChl system of LH 2 from Rhodospirillum molischianum and the peridinin-Chl a system of PCP from Amphidinium carterae. In addition, we review recent spectroscopic data for neurosporene, spheroidene and lycopene, three carotenoids with different lengths of conjugated systems. On the basis of the measured energies, emission lineshapes, solution and protein environment lifetimes for their 2A⁻ _g and 1B_u ⁺ states as well as of the theoretically determined couplings, we conclude that the transfer efficiencies from the 2A_g ⁻ state are controlled by the Car(2A_g ⁻)–BChl(Q_g) electronic couplings and the 2A_g ⁻ → 1A_g ⁻ internal conversion rates. We suggest that symmetry breaking and geometry rather than length of conjugated system dominate couplings involving the 2A_g ⁻ state. Differences in transfer efficiencies from the 1B_u ⁺ state in LH 2 and PCP are found to be dominated by the differences in spectral overlap. The role of the 1B_u ⁺ state is likely to be influenced by a lower-lying (in longer polyenes), optically forbidden 1B_u ⁻ state. This revised version was published online in June 2006 with corrections to the Cover Date.     

Splicing of Arabidopsis tRNAMet precursors in tobacco cell and wheat germ extracts

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA^Tyr _{G A} and elongator tRNA^Met _CmAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA^Tyr. Here we have studied the expression of an Arabidopsis elongator tRNA^Met gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA^Met precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA^Met to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3 and 5 splice sites and of a structured intron for pre-tRNA^Met splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA^Met splicing and that a highly structured intron is indispensable for pre-tRNA^Met splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA^Met gene, is efficiently processed and spliced in both plant extracts.     

Membranous glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1)

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-gamma. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1-/- MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1-/- MRL/lpr mice displayed significantly reduced IFN-gamma production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.