Both infiltrating regulatory T cells and insufficient antigen presentation are involved in long-term cardiac xenograft survival

We have previously shown that pretransplant donor lymphocyte infusion (DLI) together with transient depletion of CD4(+) T cells could induce permanent rat-to-mouse heart graft survival, whereas depleting CD4(+) T cells alone failed to do so. In this study, we investigated the mechanism leading to long-term xenograft survival. We found that peripheral CD4(+) T cells from DLI/anti-CD4-treated mice could mount rat heart graft rejection after adoptive transfer into B6 CD4(-/-) mice. Infusing donor-Ag-loaded mature dendritic cells (DCs) could break long-term cardiac xenograft survival in DLI/anti-CD4-treated mice. Interestingly, when the number and phenotype of graft-infiltrating cells were compared between anti-CD4- and DLI/anti-CD4-treated groups, we observed a significant increase in both the number and suppressive activity of alphabeta-TCR(+)CD3(+)CD4(-)CD8(-) double negative regulatory T cells and decrease in the numbers of CD4(+) and CD8(+) T cells in the xenografts of DLI/anti-CD4-treated mice. Moreover, there was a significant reduction in MHC class II-high DCs within the xenografts of DLI/anti-CD4-treated recipients. DCs isolated from the xenografts of anti-CD4- but not DLI/anti-CD4-treated recipients could stimulate CD4(+) T cell proliferation. Our data indicate that functional anti-donor T cells are present in the secondary lymphoid organs of the mice that permanently accepted cardiac xenografts. Their failure to reject xenografts is associated with an increase in double negative regulatory T cells as well as a reduction in Ag stimulation by DCs found within grafts. These findings suggest that local regulatory mechanisms need to be taken into account to control anti-xenograft T cell responses.     

Lysigenous aerenchyma formation in Arabidopsis is controlled by LESION SIMULATING DISEASE1

Aerenchyma tissues form gas-conducting tubes that provide roots with oxygen under hypoxic conditions. Although aerenchyma have received considerable attention in Zea mays, the signaling events and genes controlling aerenchyma induction remain elusive. Here, we show that Arabidopsis thaliana hypocotyls form lysigenous aerenchyma in response to hypoxia and that this process involves H(2)O(2) and ethylene signaling. By studying Arabidopsis mutants that are deregulated for excess light acclimation, cell death, and defense responses, we find that the formation of lysigenous aerenchyma depends on the plant defense regulators LESION SIMULATING DISEASE1 (LSD1), ENHANCED DISEASE SUSCEPIBILITY1 (EDS1), and PHYTOALEXIN DEFICIENT4 (PAD4) that operate upstream of ethylene and reactive oxygen species production. The obtained results indicate that programmed cell death of lysigenous aerenchyma in hypocotyls occurs in a similar but independent manner from the foliar programmed cell death. Thus, the induction of aerenchyma is subject to a genetic and tissue-specific program. The data lead us to conclude that the balanced activities of LSD1, EDS1, and PAD4 regulate lysigenous aerenchyma formation in response to hypoxia.     

Expression of proopiomelanocortin, proenkephalin and prodynorphin genes in porcine theca and granulosa cells

Previous studies have demonstrated the presence of endogenous opioid peptides (EOP) in the ovary and suggested their implication in local interactions within ovarian structures. Nevertheless, data pertaining to the expression of genes, coding for the opioid precursors, in ovarian cells are still rudimentary and not available for the pig. The study was undertaken to test whether genes of the opioid precursors - proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN) - are expressed in non-treated and gonadotropin-treated theca and granulosa cells isolated from ovarian follicles of the pig. The cells were isolated from small (days 15-16 of the estrous cycle) and large (days 19-20) porcine follicles. Dispersed cells were cultured in Eagle's medium under the water saturated atmosphere of 95% air and 5% CO(2), in the presence or absence of respective gonadotropin; theca cells with LH (100 ng/ml) and granulosa cells with FSH (100 ng/ml). Following 24h-incubation, the cells were harvested and the total RNA was isolated. The expression of genes coding for opioid precursors was estimated by the semi-quantitative RT-PCR technique involving co-amplification of the target cDNA (POMC, PENK or PDYN) and control cDNA (beta-actin or 18S rRNA). Specificities of PCR products were confirmed by Southern analysis and sequencing. In theca cells the expression of opioid precursors appeared to be gonadotropin-dependent except for PENK in the cells isolated from large follicles. In turn, granulosa cells exhibited the expression of POMC and PENK genes independently on treatment with FSH. This gonadotropin induced the expression of PDYN gene in granulosa cells isolated from small and large follicles and significantly increased POMC mRNA content in the cells from the large ones. The present studies indicate that porcine follicular cells (especially granulosa cells) may produce opioid peptides and that gonadotropins may modulate gene expression of their precursors in these cells. Moreover, our results support a participation of opioid peptides in the local regulations within ovarian follicle.     

Effect of norflurazon on resorcinolic lipid metabolism in rye seedlings

Norflurazon is a selective pyridazinone herbicide excessively employed in the control of many annual grasses and broad-leaved weeds. This chemical causes plant bleaching due to the inhibition of the carotenoid pigment biogenesis as well as induces irreparable changes to chloroplasts, which are considered the organelles where the biosynthesis of resorcinolic lipids takes place. Resorcinolic lipids, a group of phenolic compounds, constitute not only an essential part of the plant antifungal defense system, but also are an important component of the human cereal diet. The aim of this study was to investigate the effect of norflurazon on the biosynthesis of resorcinolic lipids in 5-day-old rye plants (Secale cereale L.) that were grown at three different temperatures under light or dark conditions. At all tested temperatures, norflurazon decreased the fresh biomass of light-grown rye seedlings and increased the weight of plants grown in darkness. Compared with respective controls, this herbicide caused an increase in total content of alkylresorcinols in both green and etiolated plants with the exception of dark-grown norflurazon-treated rye at 29 degrees C. The general level of saturated homologues was markedly decreased by norflurazon in all etiolated plants and in light-grown seedlings at 15 degrees C. Independent of thermal and light conditions, in all norflurazon-treated samples two alkylresorcinol derivatives predominated: 1,3-dihydroxy-5-n-heptadecylbenzene and 1,3-dihydroxy-5-n-nonadecylbenzene. Thus, our results suggest that norflurazon affected the metabolism of alkylresorcinols in rye seedlings and its action was dependent on external stimuli.     

Analysis of 5-methylcytosine in DNA of breast and colon cancer tissues

5-methylcytosine (m(5)C) can be used as a sensitive marker of progress of the tumor formation induced by the oxidative damage reactions. We have analyzed the amount of m(5)C in DNA of patients with breast and colon cancers. Two dimensional thin layer chromatography (TLC) has been used to monitor 5-methylcytosine level in DNA extracted from cancer tissues. The level of methylation of cytosine at C-5 position in DNA from breast cancer patients correlates well with the malignancy of tumors. Interestingly higher amount of m(5)C in DNA for the breast cancer patients treated with different chemotherapeutics was observed. It suggests an activation of DNA methyltransferase as well as a genomic suppression of the DNA repair genes expression. These differences clearly reflect the health condition of patients and support the global analysis of m(5)C in DNA as a good marker for diagnosis of neoplasia in clinical practice.     

Benzotriazinone and benzopyrimidinone derivatives as potent positive allosteric AMPA receptor modulators

AMPA receptors (AMPARs) have been demonstrated to be an important therapeutic CNS target. A series of substituted benzotriazinone and benzopyrimidinone derivatives were prepared with the aim to improve in vivo activity over the previously reported bis-benzoxazinone based AMPAKINE series from our laboratory. These compounds were shown to be potent, positive allosteric AMPAR modulators that have better in vivo activity and improved metabolic stability over the analogous benzoxazinone derivatives.     

Inhibition of LuxS by S-ribosylhomocysteine analogues containing a [4-aza]ribose ring

LuxS (S-ribosylhomocysteinase) catalyzes the cleavage of the thioether linkage of S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor to a small signaling molecule that mediates interspecies bacterial communication called autoinducer 2 (AI-2). Inhibitors of LuxS should interfere with bacterial interspecies communication and potentially provide a novel class of antibacterial agents. In this work, SRH analogues containing substitution of a nitrogen atom for the endocyclic oxygen as well as various deoxyriboses were synthesized and evaluated for LuxS inhibition. Two of the [4-aza]SRH analogues showed modest competitive inhibition (K(I) ～40 μM), while most of the others were inactive. One compound that contains a hemiaminal moiety exhibited time-dependent inhibition, consistent with enzyme-catalyzed ring opening and conversion into a more potent species (K(I)(?)=3.5 μM). The structure-activity relationship of the designed inhibitors highlights the importance of both the homocysteine and ribose moieties for high-affinity binding to LuxS active site.     

AutoMotif server: prediction of single residue post-translational modifications in proteins

The AutoMotif Server allows for identification of post-translational modification (PTM) sites in proteins based only on local sequence information. The local sequence preferences of short segments around PTM residues are described here as linear functional motifs (LFMs). Sequence models for all types of PTMs are trained by support vector machine on short-sequence fragments of proteins in the current release of Swiss-Prot database (phosphorylation by various protein kinases, sulfation, acetylation, methylation, amidation, etc.). The accuracy of the identification is estimated using the standard leave-one-out procedure. The sensitivities for all types of short LFMs are in the range of 70%. AVAILABILITY: The AutoMotif Server is available free for academic use at http://automotif.bioinfo.pl/     

Possible role of factor XIII subunit A in Fcgamma and complement receptor-mediated phagocytosis

Besides its traditional role in hemostasis, factor XIII subunit A (FXIII-A) is supposed to function as a cellular transglutaminase and to be involved in certain intracellular processes, including cytoskeletal remodeling. To investigate its intracellular role, the aim of the present study was to follow changes in FXIII-A production in combination with the receptor-mediated phagocytic activities of monocytes/macrophages and to examine the phagocytic functions of monocytes in patients with FXIII-A deficiency. Human blood monocytes were isolated from the buffy coats of healthy volunteers and cultured for 4 days. The FcgammaR-mediated phagocytosis of sensitized erythrocytes (EA) and the complement receptor (CR)-mediated phagocytosis of complement-coated yeast particles were studied during monocyte/macrophage differentiation. Changes in the gene expression of FXIII-A were detected by real-time quantitative RT-PCR. FXIII-A protein production was investigated with fluorescent image analysis at single cell level and Western immunoblot analysis. Both the FcgammaR and CR-mediated phagocytosis increased during culturing, which peaked on day 3. The phagocytic activity of the cells could be markedly inhibited with monodansylcadaverine, an inhibitor of the transglutaminase-induced crosslinking of proteins. The phagocytosis of EA, complement-coated and uncoated yeast particles was found to be strongly diminished in monocytes of FXIII-A deficient patients. The phagocytic functions of cultured cells showed a change in parallel with the alterations in FXIII-A mRNA expression, as well as with that in FXIII-A in protein synthesis detected by image and Western immunoblot analyses in concert. Our results suggest that FXIII-A plays a role in the Fcgamma and complement receptor-mediated phagocytic activities of monocytes/macrophages.     

Image processing for accurate cell recognition and count on histologic slides

OBJECTIVE: To design an automatic system for recognition and count of two different cell families on histologic slides. STUDY DESIGN: The segmentation strategy uses color information on the image. The morphologic operations and Support Vector Machine approaches are used for each color to obtain precise segmentation of the image into separate cells for recognition. RESULTS: A large set of histologic slides of bone marrow was assessed byour system and the results compared to the score of a human expert. The results are in good agreement. The difference is within acceptable limits (below 10%). CONCLUSION: The automatic system of cell recognition and extraction is accurate and provides a useful tool for cell recognition and count on histologic slides.