Fetal cells in mother rats contribute to the remodeling of liver and kidney after injury

Fetal microchimerism indicates a mixture of cells of maternal and fetal origin seen in maternal tissues during and after pregnancy. Controversy exists about whether persistent fetal microchimerism is related with some autoimmune disorders occurring during and after pregnancy. In the current experiment, an animal model in which EGFP positive cells were taken as fetal-origin cells was designed to detect the fetal microchimerism in various maternal organs. Ethanol drinking and gentamicin injection were adopted to induce liver and kidney injury simultaneously. EGFP positive cells were engrafted not only in the maternal circulation and bone marrow, but also in the liver and kidney as hepatocytes and tubular cells, respectively. These results indicate that fetal cells are engrafted to maternal hematopoietic system without apparent injury and they also contribute to the repairing process of maternal liver and kidney.     

Direct reciprocal effects of resistin and adiponectin on vascular endothelial cells: a new insight into adipocytokine-endothelial cell interactions

Resistin is an adipocytokine which plays a role in the development of insulin resistance. In this study, we investigated the direct effect of resistin on vascular endothelial cells. Resistin induced the expression of adhesion molecules such as VCAM-1 and ICAM-1, and long pentraxin 3, a marker of inflammation. The induction of VCAM-1 by resistin was inhibited partially by pitavastatin. Moreover, the induction of VCAM-1 and ICAM-1 by resistin was inhibited by adiponectin, an adipocytokine that improves insulin resistance. Taken together, these results suggest that the balance in the concentrations of adipocytokines such as resistin and adiponectin determines the inflammation status of vasculature, and in turn the progress of atherosclerosis.     

Enhancement of serum- and platelet-derived growth factor-induced cell proliferation by Necl-5/Tage4/poliovirus receptor/CD155 through the Ras-Raf-MEK-ERK signaling

Necl-5/Tage4/poliovirus receptor/CD155 has been shown to be the poliovirus receptor and to be up-regulated in rodent and human carcinoma. We have found previously that mouse Necl-5 regulates cell motility. We show here that mouse Necl-5 is furthermore involved in the regulation of cell proliferation. Studies using a specific antibody against Necl-5 and a dominant negative mutant of Necl-5 revealed that Necl-5 enhanced the serum-induced proliferation of NIH3T3, Swiss3T3, and mouse embryonic fibroblast cells. Necl-5 enhanced the serum-induced activation of the Ras-Raf-MEK-ERK signaling, up-regulated cyclins D2 and E, and down-regulated p27(Kip1), eventually shortening the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 similarly enhanced the platelet-derived growth factor-induced activation of the Ras-Raf-MEK-ERK signaling and shortened the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 acted downstream of the platelet-derived growth factor receptor and upstream of Ras. Moreover, up-regulated Necl-5 was involved at least partly in the enhanced proliferation of transformed cells including NIH3T3 cells transformed by an oncogenic Ras or v-Src. These results indicate that Necl-5 plays roles not only in cell motility but also in cell proliferation.     

Detection in situ and characterization of lignin in the<Emphasis Type="Italic"> G</Emphasis>-layer of tension wood fibres of<Emphasis Type="Italic"> Populus deltoides</Emphasis>

The occurrence of lignin in the additional gelatinous (G-) layer that differentiates in the secondary wall of hardwoods during tension wood formation has long been debated. In the present work, the ultrastructural distribution of lignin in the cell walls of normal and tension wood fibres from poplar (Populus deltoides Bartr. ex Marshall) was investigated by transmission electron microscopy using cryo-fixation–freeze-substitution in association with immunogold probes directed against typical structural motifs of lignin. The specificity of the immunological probes for condensed and non-condensed guaiacyl and syringyl interunit linkages of lignin, and their high sensitivity, allowed detection of lignin epitopes of definite chemical structures in the G-layer of tension wood fibres. Semi-quantitative distribution of the corresponding epitopes revealed the abundance of syringyl units in the G-layer. Predominating non-condensed lignin sub-structures appeared to be embedded in the crystalline cellulose matrix prevailing in the G-layer. The endwise mode of polymerization that is known to lead to these types of lignin structures appears consistent with such an organized cellulose environment. Immunochemical labelling provides the first visualization in planta of lignin structures within the G-layer of tension wood. The patterns of distribution of syringyl epitopes indicate that syringyl lignin is deposited more intensely in the later phase of fibre secondary wall assembly. The data also illustrate that syringyl lignin synthesis in tension wood fibres is under specific spatial and temporal regulation targeted differentially throughout cell wall layers.Abbreviations G-layer Gelatinous layer - G Guaiacyl monomeric unit - PATAg Periodic acid–thiocarbohydrazide–silver proteinate - S Syringyl monomeric unit     

Determination of rat blood S-D-lactoylglutathione by a column-switching high-performance liquid chromatography with precolumn fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

A method for the determination of S-d-lactoylglutathione (SLG), an intermediate metabolite of the glyoxalase system, in rat blood is described. After hemolysis and deproteinization of 30 microl of rat blood, SLG in the blood was determined by a column-switching HPLC system with precolumn fluorescence derivatization with a fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Calibration curves for the determination of SLG showed a good linearity (r2 > 0.999) over the range of 20-100 pmol SLG spiked in rat blood samples. The accuracy was in the range of 97-104% (20-100 pmol SLG spiked in rat blood sample, N = 4). The detection limit was 8.12 fmol, and the quantitation limit was 27.07 fmol, respectively. The intra- and interday coefficients of variance were 4.63% (N = 5) and 9.98% (N = 5), respectively. The concentrations of SLG in whole blood from male Wistar-Kyoto rats (12 weeks old) were 3.48+/-0.78 microM (N = 4, mean+/-SE). In streptozotocin-induced diabetic rats, the concentration of SLG was significantly increased, approx 5-fold, compared with normal rats, suggesting that the metabolic flux of the glyoxalase system increases in red blood cells during hyperglycemia.     

Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POLbeta, POLdelta, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POLbeta, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POLbeta dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).     

Analysis of major virulence factors in Porphyromonas gingivalis under various culture temperatures using specific antibodies

Porphyromonas gingivalis is implicated in the occurrence of adult periodontitis. We have previously identified major outer membrane proteins from P. gingivalis, which include representative virulence factors such as gingipains, a 75 kDa major protein, RagA, RagB, and putative porin. Fimbriae, another important virulence factor, exist on the cell surface. In this study, we identified major supernatant proteins. They were fimbrilin, the 75 kDa major protein, gingipains and their adhesin domains. Microscopic examination showed that supernatant proteins formed vesicle-like and fimbrial structures. To learn more about the character of this bacterium, we examined effects of growth temperature on localization and expression of these virulence factors. In general, localization of major virulence factors did not change at the various growth temperatures used. Most of the 75 kDa major protein, RagA, RagB, and putative porin were found in the envelope fraction, not in cell-free culture supernatant. Gingipains were found in both the envelope fraction and supernatant. More than 80% of fimbriae were associated with cells, less than 20% migrated to the supernatant. Most fimbriae existed in the whole cell lysate, although there was a small amount in the envelope fraction. When the growth temperature was increased, expression of fimbriae, gingipains, the 75 kDa major protein, RagA, and RagB decreased. However, temperature had almost no effect on expression of putative porin. The tendency for expression of major virulence factors to decrease at higher temperatures may enable P. gingivalis to survive under hostile conditions.     

Complement inhibitors targeted to the proximal tubule prevent injury in experimental nephrotic syndrome and demonstrate a key role for C5b-9

In glomerular diseases of diverse etiologies, dysfunction of the glomerular barrier to protein passage results in proteinuria, and proteinuria is considered an independent risk factor that plays a direct role in inflammation, interstitial fibrosis, and renal failure. The mechanism by which proteinuria leads to nephrotoxic injury is unclear, but a role for complement in mediating interstitial damage appears likely. We describe a strategy for Ag-specific targeting of complement inhibitors using a single chain Ab fragment and show that complement inhibitors targeted to the tubular epithelium protect against tubulointerstitial injury and renal dysfunction in a rat model of puromycin-induced nephrosis. The targeting of systemically administered complement inhibitors markedly enhanced their efficacy and obviated the need to systemically inhibit complement, thus reducing the risk of compromising host defense and immune homeostasis. Targeted inhibition of complement activation by Crry, and of membrane attack complex (MAC) formation by CD59 was equally therapeutic, demonstrating that the MAC plays a key role in proteinuria-induced tubulointerstitial injury. CD59 activity was dependent on its being targeted to the site of complement activation, and this is the first report of specific inhibition of the MAC in vivo after systemic administration of inhibitor. The data establish the MAC is a valid target for pharmaceutical intervention in proteinuric disorders and provide an approach to investigate the role of the MAC in complement-dependent disease under clinically relevant conditions.