Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Thrombospondin-1 expression and modulation of Wnt and hippo signaling pathways during the early phase of Trypanosoma cruzi infection of heart endothelial cells

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/β-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/β-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/β-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3β at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3β was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of β-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of β-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating β-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a β-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the β-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.     

Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC

Protein function prediction is very important in establishing the roles of various proteins in bacteria; however, some proteins in the E. coli genome have their function assigned based on low percent sequence homology that does not provide reliable assignments. We have made an attempt to verify the prediction that E. coli genes ygiC and yjfC encode proteins with the same function as glutathionylspermidine synthetase/amidase (GspSA). GspSA is a bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine (G-Sp), a conjugate of glutathione (GSH) and spermidine. YgiC and YjfC proteins show 51% identity between themselves and 28% identity to the synthetase domain of the GspSA enzyme. YgiC and YjfC proteins were expressed and purified, and the properties of GspSA, YgiC, and YjfC were compared. In contrast to GspSA, proteins YgiC and YjfC did not bind to G-Sp immobilized on the affinity matrix. We demonstrated that all three proteins (GspSA, YgiC and YjfC) catalyze the hydrolysis of ATP; however, YgiC and YjfC cannot synthesize G-Sp, GSH, or GSH intermediates. gsp, ygiC, and yjfC genes were eliminated from the E. coli genome to test the ability of mutant strains to synthesize G-Sp conjugate. E. coli cells deficient in GspSA do not produce G-Sp while synthesis of the conjugate is not affected in ΔygiC and ΔyjfC mutants. All together our results indicate that YgiC and YjfC are not glutathionylspermidine synthetases as predicted from the amino acid sequence analysis.     

Thrombospondin-1 interacts with Trypanosoma cruzi surface calreticulin to enhance cellular infection

Trypanosoma cruzi causes Chagas disease, which is a neglected tropical disease that produces severe pathology and mortality. The mechanisms by which the parasite invades cells are not well elucidated. We recently reported that T. cruzi up-regulates the expression of thrombospondin-1 (TSP-1) to enhance the process of cellular invasion. Here we characterize a novel TSP-1 interaction with T. cruzi that enhances cellular infection. We show that labeled TSP-1 interacts specifically with the surface of T. cruzi trypomastigotes. We used TSP-1 to pull down interacting parasite surface proteins that were identified by mass spectrometry. We also show that full length TSP-1 and the N-terminal domain of TSP-1 (NTSP) interact with T. cruzi surface calreticulin (TcCRT) and other surface proteins. Pre-exposure of recombinant NTSP or TSP-1 to T. cruzi significantly enhances cellular infection of wild type mouse embryo fibroblasts (MEF) compared to the C-terminal domain of TSP-1, E3T3C1. In addition, blocking TcCRT with antibodies significantly inhibits the enhancement of cellular infection mediated by the TcCRT-TSP-1 interaction. Taken together, our findings indicate that TSP-1 interacts with TcCRT on the surface of T. cruzi through the NTSP domain and that this interaction enhances cellular infection. Thus surface TcCRT is a virulent factor that enhances the pathogenesis of T. cruzi infection through TSP-1, which is up-regulated by the parasite.     

Diaphragm adaptations in patients with COPD

Inspiratory muscle weakness in patients with COPD is of major clinical relevance. For instance, maximum inspiratory pressure generation is an independent determinant of survival in severe COPD. Traditionally, inspiratory muscle weakness has been ascribed to hyperinflation-induced diaphragm shortening. However, more recently, invasive evaluation of diaphragm contractile function, structure, and biochemistry demonstrated that cellular and molecular alterations occur, of which several can be considered pathologic of nature. Whereas the fiber type shift towards oxidative type I fibers in COPD diaphragm is regarded beneficial, rendering the overloaded diaphragm more resistant to fatigue, the reduction of diaphragm fiber force generation in vitro likely contributes to diaphragm weakness. The reduced diaphragm force generation at single fiber level is associated with loss of myosin content in these fibers. Moreover, the diaphragm in COPD is exposed to oxidative stress and sarcomeric injury. This review postulates that the oxidative stress and sarcomeric injury activate proteolytic machinery, leading to contractile protein wasting and, consequently, loss of force generating capacity of diaphragm fibers in patients with COPD. Interestingly, several of these presumed pathologic alterations are already present early in the course of the disease (GOLD I/II), although these patients appear not limited in their daily life activities. Treatment of diaphragm dysfunction in COPD is complex since its etiology is unclear, but recent findings indicate the ubiquitin-proteasome pathway as a prime target to attenuate diaphragm wasting in COPD.     

增强UV-B辐射对蚕豆叶片微粒体膜一些性质的影响

温室条件下,用0（Control）、8．65kJm－2d－1（TI）及11．2KJm－2d－1（t2）不同剂量的UV－B辐射处理蚕豆幼苗。Ca2 ．ATPase及Mg2 －ATPase的活性在辐射处理期间下降。在处理21d,T1和T2微粒体膜的MDA含量明显高于对照,同时IUFA急剧下降,且呈明显的剂量效应。14及21d时,膜磷脂的含量也明显下降。脂氧合酶（Lox）活性在第7及14天与对照相比都显著升高,而21d后迅速下降。结果表明,增强UV－B对微粒体膜的伤害可能是一方面导致正常酶合成与分解之间的平衡失调,另一方面导致了膜脂过氧化作用。