Intracellular survival of Shigella

Bacterial invasion of eukaryotic cells and host recognition and killing of the invading bacteria are a key issue in determining the fate of bacterial infection. Once inside host cells, pathogenic bacteria often modify the phagosomal compartment or enter the host cytosol to escape from the lytic compartment and gain a replicative niche. Cytosolic invaders, however, are monitored by host innate immune systems, such as mediated by Nod/CARD family proteins, which induce inflammatory responses via activation of NF-kappaB. Furthermore, recent studies indicate that autophagy, a major cytoplasmic degradation system that eliminates cytosolic protein and organelles, also recognizes invading bacteria. Indeed, unless they are able to circumvent entrapping by autophagic membranes, bacteria targeted by autophagy ultimately undergo degradation by delivery into autolysosomes. In this article, we review recent advances in understanding of Shigella strategies to infect epithelial cells, and then focus on recent studies of an intriguing bacterial survival strategy against autophagic degradation.     

Latent adenoviral infection induces production of growth factors relevant to airway remodeling in COPD

Previous studies showed an association between latent adenoviral infection with expression of the adenoviral E1A gene and chronic obstructive pulmonary disease (COPD). The present study focuses on how the adenoviral E1A gene could alter expression of growth factors by human bronchial epithelial (HBE) cells. The data show that connective tissue growth factor (CTGF) and transforming growth factor (TGF)-beta 1 mRNA and protein expression were upregulated in E1A-positive HBE cells. Upregulation of CTGF in this in vitro model was independent of TGF-beta secreted into the growth medium. Comparison of E1A-positive with E1A-negative HBE cells showed that both expressed cytokeratin but only E1A-positive cells expressed the mesenchymal markers vimentin and alpha-smooth muscle actin. We conclude that latent infection of epithelial cells by adenovirus E1A could contribute to airway remodeling in COPD by the viral E1A gene, inducing TGF-beta 1 and CTGF expression and shifting cells to a more mesenchymal phenotype.     

Proton nuclear magnetic resonance spectra of compounds I and II of horseradish peroxidase

Enzymatic reaction intermediates of horseradish peroxidase, compounds I and II, were studied by high-resolution nuclear magnetic resonance spectroscopy at 220 MHz. The heme peripheral proton peaks were successfully obtained in the downfield region of 50 to 80 ppm from 4,4-dimethyl-4-silapentane-5-sulfonate for compound I and of 10 to 20 ppm for compound II at pH 9.2. This indicates that no isoporphyrin appears in the catalytic cycle of the enzyme. Temperature dependences of the spectra also were determined for these compounds between 7 and 32 degrees C. With increasing temperature, all the peaks in the downfield region for compound I shifted upfield, obeying the Curie law. These results suggest that the Fe atoms in compounds I and II are in ferryl high- and low-spin states, respectively. The spectrum was also observed in solutions of horse metmyoglobin to which hydrogen peroxide (H2O2) was added. The electron formulations of the hemes in their spectra. Evidence was found against a pi-cation radical on the heme ring as a source of the oxidizing equivalent in compound I.     

Analysis of CpG islands of trophoblast giant cells by restriction landmark genomic scanning

Rat trophoblast giant cells each contain at least 100 times more genomic DNA per nucleus than diploid cells. This unusual phenomenon appears to be of interest in relation to the molecular mechanism of cell differentiation and gene expression in the placenta. In the present study, we analyzed the CpG islands of trophoblast giant cells by restriction landmark genomic scanning (RLGS) using the methylation-sensitive landmark enzymes, Not I and Bss HII. More than 1,000 and 1,900 spots were detected by RLGS using Not I and Bss HII, respectively, in the placental junctional zone, where more than 90% of genomic DNA is present in the cells with higher DNA content. Of these, 97% (1,009 spots) and 99% (1,911 spots) of the spots found in the junctional zone showed an identical pattern and identical intensity with those of diploid cell controls, for which genomic DNA was extracted from the labyrinth zone and maternal kidney. Therefore, the giant cells are basically polyploid. More importantly, 24 tissue-specific spots were detected by RLGS using Not I. Subsequent cloning and sequencing of four typical spots of the genomic DNA confirmed that these DNA fragments contained abundant CpG dinucleotides and showed characteristics of CpG islands. Of these 24 spots, there were ten spots specific for the placenta, and three of them were specific for the junctional zone, indicating that methylation status of CpG islands in the placental tissue differed between the junctional zone and labyrinth zone. These results suggest that multiple rounds of endoreduplication and modification of CpG islands by cytosine methylation occur during the differentiation process of giant cells. Dev. Genet. 22:132–140, 1998. © 1998 Wiley-Liss, Inc.     

Role of phosphoenolpyruvate in the NADP-isocitrate dehydrogenase and isocitrate lyase reaction in Escherichia coli

Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki' of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.     

Thrombin regulates the function of human blood dendritic cells

Thrombin is the key enzyme in the coagulation cascade and activates endothelial cells, neutrophils and monocytes via protease-activated receptors (PARs). At the inflammatory site, immune cells have an opportunity to encounter thrombin. However little is known about the effect of thrombin for dendritic cells (DC), which are efficient antigen-presenting cells and play important roles in initiating and regulating immune responses. The present study revealed that thrombin has the ability to stimulate blood DC. Plasmacytoid DC (PDC) and myeloid DC (MDC) isolated from PBMC expressed PAR-1 and released MCP-1, IL-10, and IL-12 after thrombin stimulation. Unlike blood DC, monocyte-derived DC (MoDC), differentiated in vitro did not express PAR-1 and were unresponsive to thrombin. Effects of thrombin on blood DC were significantly diminished by the addition of anti-PAR-1 Ab or hirudin, serine protease inhibitor. Moreover, thrombin induced HLA-DR and CD86 expression on DC and the thrombin-treated DC induced allogenic T cell proliferation. These findings indicate that thrombin plays a role in the regulation of blood DC functions.     

Rheological characteristics of heat-induced custard pudding gels with high antioxidative activity

Custard pudding gels were prepared from fresh whole egg, milk and sugar. The effects of D-psicose (Psi), a non-calorie rare hexose, on the antioxidative activity and rheological properties of the custard pudding gels were investigated at different temperatures for comparison with those of control sugars (sucrose, Suc; D-fructose, Fru). The rheological behavior of the heat-induced pudding gels was evaluated by using breaking and creep tests. During the heat-induced gel formation, custard pudding containing Psi (15%, wt/wt) demonstrated a stronger breaking strength and higher viscoelasticity than those containing Fru and Suc. The thermodynamic parameters obtained from DSC indicated that the egg white (EW) proteins were made less thermally stable when heated in the presence of Psi than in the presence of Fru and Suc. These findings are consistent with enhanced aggregation of the EW solution in the presence of Psi. Furthermore, the Psi pudding gels possessed higher antioxidative activity than the control sugar pudding gels by using an analysis of the scavenging activity on DPPH radicals and the ferric-reducing antioxidative power. These results suggest that Psi favored cross-linking of Psi-protein molecules through the Maillard reaction which increased the formation of intermediate products to improve functionality. Custard pudding containing Psi could therefore be an effective functional sweet desert with high antioxidative activity and the outstanding gelling characteristics.     

Treatment of murine SCC VII tumors with localized hyperthermia and temperature-sensitive liposomes containing cisplatin

The release of cisplatin (CDDP) encapsulated in temperature-sensitive unilamellar liposomes to murine SCC VII carcinoma by localized hyperthermia and the effects of the treatment on tumor growth were studied. A transition temperature of the temperature-sensitive liposomes containing cisplatin (LIP-CDDP) was 41 degrees C. Twenty-four hours after injection of LIP-CDDP, the heated tumors (42 degrees C, 60 min) contained 3.3 times more CDDP than the unheated tumors receiving free CDDP. Although the uptake of liposome-associated CDDP by liver was approximately threefold greater at 1.5 h after injection than uptake of free CDDP, it decreased about 50% over a 24-h period. No difference in uptake of the two forms of CDDP by kidney was observed. The combination of LIP-CDDP and localized heating at 42 or 43 degrees C was more effective relative to the amount of CDDP in delaying tumor growth than that of free CDDP and hyperthermia. Treatment with LIP-CDDP plus local heating resulted in a dose-modifying factor of 5.3 when compared with free CDDP and no hyperthermia. The dose-modifying factor was 2.8 when treatment with LIP-CDDP and heat was compared with treatment with free CDDP and heat. Thus CDDP could be released selectively from the temperature-sensitive liposomes by heat and resulted in both a greater uptake of the drug and a delay in tumor growth.     

Luteolin and Quercetin Affect the Cholesterol Absorption Mediated by Epithelial Cholesterol Transporter Niemann–Pick C1-Like 1 in Caco-2 Cells and Rats

Niemann–Pick C1-Like 1 (NPC1L1) mediates cholesterol absorption, and ezetimibe is a potent NPC1L1 inhibitor applicable for medication of hypercholesterolemia. Epidemiological studies demonstrated that consumption of polyphenols correlates with a decreased risk for atherosclerosis due to their antioxidant effect. This activity can hardly be attributable to the antioxidant activity only, and we hypothesized that polyphenols inhibit intestinal transport of cholesterol. We elucidated the kinetic parameters of intestinal cholesterol absorption, screened several polyphenols for their ability to specifically inhibit intestinal cholesterol absorption, and determined the inhibitory effects of selected flavonoids in vitro and in vivo. The concentration-dependent uptake of cholesterol by Caco-2 cells obeyed a monophasic saturation process. This indicates the involvement of an active-passive transport, i.e., NPC1L1. Parameters of cholesterol uptake by Caco-2 cells were as follows: J _max, K _t, and K _d were 6.89±2.96 19.03±11.58 µM, and 0.11±0.02 pmol/min/mg protein, respectively. Luteolin and quercetin inhibited cholesterol absorption by Caco-2 cells and human embryonic kidney 293T cells expressing NPC1L1. When preincubated Caco-2 cells with luteolin and quercetin before the assay, cholesterol uptake significantly decreased. The inhibitory effects of these flavonoids were maintained for up to 120 min. The level of inhibition and irreversible effects were similar to that of ezetimibe. Serum cholesterol levels significantly decreased more in rats fed both cholesterol and luteolin (or quercetin), than in those observed in the cholesterol feeding group. As quercetin induced a significant decrease in the levels of NPC1L1 mRNA in Caco-2 cells, the in vivo inhibitory effect may be due to the expression of NPC1L1. These results suggest that luteolin and quercetin reduce high blood cholesterol levels by specifically inhibiting intestinal cholesterol absorption mediated by NPC1L1.