Busulfan-induced senescence is dependent on ROS production upstream of the MAPK pathway

Induction of cellular senescence is a common response of a normal cell to a DNA-damaging agent, which may contribute to cancer chemotherapy- and ionizing radiation-induced normal tissue injury. The induction has been largely attributed to the activation of p53. However, the results from the present study suggest that busulfan (BU), an alkylating agent that causes DNA damage by cross-linking DNAs and DNA and proteins, induces senescence in normal human diploid WI38 fibroblasts through the extracellular signal-regulated kinase (Erk) and p38 mitogen-activated protein kinase (p38 MAPK) cascade independent of the p53-DNA damage pathway. The induction of WI38 cell senescence is initiated by a transient depletion of intracellular glutathione (GSH) and followed by a continuous increase in reactive oxygen species (ROS) production via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which leads to the activation of the Erk and p38 MAPK pathway. Incubation of WI38 cells with N-acetylcysteine (NAC) replenishes intracellular GSH, abrogates the increased production of ROS, ameliorates Erk and p38 MAPK activation, and attenuates senescence induction by BU. Thus, inhibition of senescence induction using a potent antioxidant or specific inhibitor of the Erk and p38 MAPK pathway has the potential to be developed as a mechanism-based strategy to ameliorate cancer therapy-induced normal tissue damage.     

Cytokine-induced upregulation of NF-kappaB, IL-8, and ICAM-1 is dependent on colonic cell polarity: implication for PKCdelta

As described for a long time, carcinoma-derived Caco-2 cells form a polarized epithelium in culture, whereas HT29-D4 cells are nonpolarized and undifferentiated but can form a polarized monolayer when cultured in a galactose-supplemented medium. Using NF-kappaB translocation and IL-8 and ICAM-1 gene activation as an index, we have studied the relationship between the differentiation state and the cell response to cytokines. We found that differentiated Caco-2 and HT29-D4 cells were responsive to both cytokines TNFalpha- and IL-1beta-mediated activation of NF-kappaB but that undifferentiated HT29-D4 cells were unresponsive to IL-1beta. However, the expression of endogenous ICAM-1 and IL-8 genes was upregulated by these cytokines in either cell lines differentiated or not. Upregulation of ICAM-1 gene occurred when IL-1beta or TNFalpha was added to the basal, but not apical surface of the differentiated epithelia. Finally, it appeared that in polarized HT29-D4 cells, the IL-1beta-induced translocation of NF-kappaB was connected to PKCdelta translocation.     

The phosphorylation state of MRLC is polyamine dependent in intestinal epithelial cells

Cell migration is important to the integrity of the gastrointestinal tract for the normal movement of cells from crypt to villi and the healing of wounds. Polyamines are essential to cell migration, mucosal restitution, and, hence, healing. Polyamine depletion by α-difluoromethyl ornithine (DFMO) inhibited migration by decreasing lamellipodia and stress fiber formation and preventing the activation of Rho-GTPases. Polyamine depletion increased the association of the thick F-actin cortex with phosphorylated myosin regulatory light chain (pMRLC). In this study, we determined why MRLC is constitutively phosphorylated as part of the actin cortex. Inhibition of myosin light chain kinase (MLCK) decreased RhoA and Rac1 activities and significantly inhibited migration. Polyamine depletion increased phosphorylation of MRLC (Thr18/Ser19) and stabilized the actin cortex and focal adhesions. The Rho-kinase inhibitor Y27632 increased spreading and migration by decreasing the phosphorylation of MRLC, remodeling focal adhesions, and by activating Rho-GTPases. Thus phosphorylation of MRLC appears to be the rate-limiting step during the migration of IEC-6 cells. In addition, increased localization of RhoA with the actin cortex in polyamine-depleted cells appears to activate Rho-kinase. In the absence of polyamines, activated Rho-kinase phosphorylates myosin phosphatase targeting subunit 1 (MYPT1) at serine-668 leading to its inactivation and preventing the recruitment of phosphatase (protein phosphastase, PP1cδ) to the actomyosin cortex. In this condition, MRLC is constitutively phosphorylated and cycling does not occur. Thus activated myosin binds F-actin stress fibers and prevents focal adhesion turnover, Rho-GTPase activation, and the remodeling of the cytoskeleton required for migration.     

Kinetic modelling of lactate utilization and butyrate production by key human colonic bacterial species

Butyrate is the preferred energy source for colonocytes and has an important role in gut health; in contrast, accumulation of high concentrations of lactate is detrimental to gut health. The major butyrate-producing bacterial species in the human colon belong to the Firmicutes. Eubacterium hallii and a new species, Anaerostipes coli SS2/1, members of clostridial cluster XIVa, are able to utilize lactate and acetate via the butyryl CoA : acetate CoA transferase route, the main metabolic pathway for butyrate synthesis in the human colon. Here we provide a mathematical model to analyse the production of butyrate by lactate-utilizing bacteria from the human colon. The model is an aggregated representation of the fermentation pathway. The parameters of the model were estimated using total least squares and maximum likelihood, based on in vitro experimental data with E. hallii L2-7 and A. coli SS2/1. The findings of the mathematical model adequately match those from the bacterial batch culture experiments. Such an in silico approach should provide insight into carbohydrate fermentation and short-chain fatty acid cross-feeding by dominant species of the human colonic microbiota.     

Hepatic steatosis in leptin-deficient mice is promoted by the PPARgamma target gene Fsp27

Peroxisome proliferator-activated receptor gamma (PPARgamma) is induced in leptin-deficient (ob/ob) mouse liver and is critical for the development of hepatic steatosis. The present study shows that fat-specific protein 27 (Fsp27) in ob/ob liver is a direct target gene of PPARgamma and can elevate hepatic triglyceride levels. FSP27 belongs to the CIDE family, composed of CIDE A, CIDE B, and FSP27/CIDE C, all of which contain a conserved CIDE-N domain. FSP27 was recently reported to be a lipid droplet-binding protein and to promote lipid accumulation in adipocytes. The Fsp27 gene was expressed at high levels in ob/ob liver and at markedly lower levels in ob/ob livers lacking PPARgamma. Forced expression of FSP27 by adenovirus in hepatocytes in vitro or in vivo led to increased triglyceride levels. Knockdown by adenovirus expressing FSP27 shRNA resulted in lower accumulation of hepatic triglycerides compared to control adenovirus-infected liver. Taken together, these results indicate that FSP27 is a direct mediator of PPARgamma-dependent hepatic steatosis.     

Vascular injury response in mice is dependent on genetic background

Mouse models are employed to unravel the pathophysiology of vascular restenosis. Although much effort has been spent on how to apply an adequate arterial injury, the influence of the genetic background of mice has not yet received sufficient consideration. The study presented herein was designed to demonstrate the influence of the mouse strain on vascular injury response. Mice of a defined background (50% 129 strain and 50% DBA strain) were backcrossed into either the 129 strain or the DBA strain. Male offspring were subjected to a femoral artery injury model by applying an electric current. Morphometric analysis revealed that backcrossing into the 129 strain resulted in a significant (P < 0.001) 17-fold increase in neointima formation (n = 17 mice) compared with backcrossing into the DBA strain (n = 19). The values of neointima area were 9.18 x 10(3) +/- 2.13 x 10(3) and 0.54 x 10(3) +/- 0.39 x 10(3) microm2, respectively. In conjunction, the vessel wall area was enhanced by 1.8-fold (P < 0.001). In contrast, no significant differences were found for the areas of the lumen and the tunica media. Similarly, a significant increase in neointima formation was also found for mice of pure 129 strain compared with pure DBA strain. The results underline the importance of the genetic background for studies on vascular injury response. Furthermore, because the mouse genome of the various strains is well defined, serial testing of the genetic background of mice will provide candidate genes and/or genetic modifiers controlling vascular injury response.     

The senescence of oat leaf segments is promoted under simulated microgravity condition on a three-dimensional clinostat.

Plants have evolved on the earth, indicating the morphology, growth and development, and life cycle of plants are highly influenced by gravity as well as other environmental stimuli. Indeed, simulated microgravity on a clinostat or hypergravity on a centrifuge has recently been reported to change the growth and development of plants (Hoson et al. 1992, 1993, 1995, Rasmussen et al. 1994, Kasahara et al. 1995). Senescence is a final drastic phenomenon in life cycle of plants, which is characterized by the loss of total chlorophyll and protein, and/or the formation of the abscission (Osborne 1973, Thimann 1977, Addicott 1982). Many environmental stimuli as well as the qualitative and quantitative changes of plant hormones have been reported to affect plant senescence. Among those stimuli, light is the most important factor to regulate plant senescence (Leopold 1964). Dark condition promotes leaf senescence due to the decrease in endogenous level of cytokinin and/or the increase in that of abscisic acid or ethylene (Tetley and Thimann 1974, Gepstein and Thimann 1980). However, there are few reports concerning the effect of gravity on leaf senescence. Strenuous effort to learn leaf senescence under microgravity condition has been done using a three-dimensional (3-D) clinostat. In this paper, we report that simulated microgravity condition on a 3-D clinostat promoted the senescence of oat leaf segments in the dark. A possible mechanism of microgravity condition on promoting the senescence is also discussed.     

Augmented lipopolysaccharide-induced TNF-alpha production by peritoneal macrophages in type 2 diabetic mice is dependent on elevated glucose and requires p38 MAPK

Dysregulated inflammation is a complication of type 2 diabetes (T2D). In this study, we show that augmented LPS-induced TNF-alpha production by resident peritoneal macrophages (PerMphi) in type 2 diabetic (db/db) mice is dependent on elevated glucose and requires p38 MAPK. Intraperitoneal LPS administered to db/db and nondiabetic (db/+) mice induced 3- and 4-fold more TNF-alpha in the peritoneum and serum, respectively, of db/db mice as compared with db/+ mice. Examination of the TLR-4/MD2 complex and CD14 expression showed no difference between db/db and db/+ PerMphi. Ex vivo stimulation of PerMphi with LPS produced a similar 3-fold increase in TNF-alpha production in db/db PerMphi when compared with db/+ PerMphi. PerMphi isolated from db/+ mice incubated in high glucose (4 g/L) medium for 12 h produced nearly 2-fold more TNF-alpha in response to LPS than PerMphi incubated in normal glucose medium (1 g/L). LPS-dependent stimulation of PI3K activity, ERK1/2 activation, and p38 kinase activity was greater in PerMphi from db/db mice as compared with db/+ mice. Only inhibition of p38 kinase blocked LPS-induced TNF-alpha production in PerMphi from db/db mice. Taken together, these data indicate that augmented TNF-alpha production induced by LPS in macrophages during diabetes is due to hyperglycemia and increased LPS-dependent activation of p38 kinase.     

Consumption of Bifidobacterium lactis LKM512 yogurt reduces gut mutagenicity by increasing gut polyamine contents in healthy adult subjects

The possible role of probiotic metabolites on human health effects of probiotics has received little research attention. In this study, we investigated the effects of consumption of Bifidobacterium lactis LKM512-containing yogurt (LKM512 yogurt) on fecal probiotic metabolites (polyamines, lactate, and acetate) and mutagenicity in seven healthy adults (one male and six females; average age: 30.5 years). Each volunteer was provided with 100g/day of LKM512 yogurt or placebo for 2 weeks. Fecal polyamines and mutagenicity were measured by HPLC and the umu-test, respectively. Consumption of LKM512 yogurt increased fecal spermidine levels, but not fecal lactate and acetate contents. The mutagenicity level significantly reduced to 79.2% (10-91.1%) and 47.9% (0-86.8%) following consumption of LKM512 yogurt (P=0.0293) and placebo (P=0.0314), respectively. LKM512 yogurt consumption significantly reduced the mutagenicity level compared with consumption of a placebo (P=0.0489). These results suggest that increased gut spermidine level by LKM512 yogurt was responsible for the reduction of mutagenicity in the gut of healthy adults. We suggest that spermidine produced by LKM512 yogurt consumption contributes to host health as a bioantimutagenic factor; to our knowledge, these substances have not been previously reported as antimutagens from probiotics or fermented milk.     

A potential role of GW4064 to inhibit gut bacterial overgrowth by activating FXR in suppression of ethanol-induced liver injury

Hepatic parenchymal and nonparenchymal cells are highly susceptible to ethanol and its metabolites, and excessive alcohol consumption results in damage to the liver. Ethanol induces an increased prevalence for bacterial overgrowth in the small intestine and translocation of endotoxin into the portal blood. Some studies have pointed to a role for activation of Kupffer cells by gut bacteria-derived endotoxin as a primary event in mechanisms of alcoholic liver injury (ALD). GW4064, a potent farnesoid X receptor (FXR) agonist, has been developed as a hepatoprotective agent, and has been used in animal models of a variety of liver diseases. At the same time, previous experimental results showed that BAs and GW4064 inhibit bacterial overgrowth in the small intestine. It is logical to postulate that GW4064 may control or alleviate the ethanol-induced liver injury through inhibiting gut bacterial overgrowth. GW4064 activates FXR, which induces the expression of several genes with potential functions in mucosal defense to prevent intestinal bacteria overgrowth and translocation into the circulation induced by ethanol, and then will alleviate ethanol-induced liver injury. The hypothesis will provide the brand-new direction that we may prevent and treat ALD by using GW4064 through activating FXR to control gut bacteria overgrowth.     

Cell-to-cell movement of potato potexvirus X is dependent on suppression of RNA silencing

RNA silencing in transgenic and virus-infected plants involves a mobile silencing signal that can move cell-to-cell and systemically through the plant. It is thought that this signal can influence long-distance movement of viruses because protein suppressors of silencing encoded in viral genomes are required for long-distance virus movement. However, until now, it was not known whether the mobile signal could also influence short-range virus movement between cells. Here, through random mutation analysis of the Potato Potexvirus X (PVX) silencing suppressor P25, we provide evidence that it does. All mutants that were defective for silencing suppression were also non-functional in viral cell-to-cell movement. However, we identified mutant P25 proteins that were functional as silencing suppressors but not as movement proteins and we conclude that suppression of silencing is not sufficient to allow virus movement between cells: there must be a second P25 function that is independent of silencing but also required for cell-to-cell movement. Consistent with this hypothesis, we identified two classes of suppressor-inactive P25 mutants. One class of these mutants is proposed to be functional for the accessory function because their failure to support PVX movement could be complemented by heterologous suppressors of silencing. The second class of P25 mutants is considered defective for both the suppressor and second functions because the heterologous silencing suppressors did not restore virus movement. It is possible, based on analyses of short interfering RNA accumulation, that P25 suppresses silencing by interfering with either assembly or function of the effector complexes of RNA silencing.     

Sepsis-associated cholestasis is critically dependent on P-selectin-dependent leukocyte recruitment in mice

Cholestasis is a major complication in sepsis although the underlying mechanisms remain elusive. The aim of this study was to evaluate the role of P-selectin and leukocyte recruitment in endotoxemia-associated cholestasis. C57BL/6 mice were challenged intraperitoneally with endotoxin (0.4 mg/kg), and 6 h later the common bile duct was cannulated for determination of bile flow and biliary excretion of bromosulfophthalein. Mice were pretreated with an anti-P-selectin antibody or an isotype-matched control antibody. Leukocyte infiltration was determined by measuring hepatic levels of myeloperoxidase. Tumor necrosis factor-alpha and CXC chemokines in the liver was determined by ELISA. Liver damage was monitored by measuring serum levels of alanine aminotransferase and aspartate aminotransferase. Apoptosis was quantified morphologically by nuclear condensation and fragmentation using Hoechst 33342 staining. Endotoxin induced a significant inflammatory response with increased TNF-alpha and CXC chemokine concentrations, leukocyte infiltration, liver enzyme release, and apoptotic cell death. This response was associated with pronounced cholestasis indicated by a >70% decrease of bile flow and biliary excretion of bromosulfophthalein. Immunoneutralization of P-selectin significantly attenuated endotoxin-induced leukocyte infiltration reflected by a >60% reduction of hepatic myeloperoxidase levels. Interference with P-selectin decreased endotoxin-mediated hepatocellular apoptosis and necrosis, but did not affect hepatic levels of tumor necrosis factor-alpha and CXC chemokines. Of interest, inhibition of P-selectin restored bile flow and biliary excretion of bromosulfophthalein to normal levels in endotoxin-challenged animals. Our study demonstrates for the first time that P-selectin-mediated recruitment of leukocytes, but not the local production of proinflammatory mediators, is the primary cause of cholestasis in septic liver injury.     

Alternative spermidine biosynthetic route is critical for growth of Campylobacter jejuni and is the dominant polyamine pathway in human gut microbiota

The availability of fully sequenced bacterial genomes has revealed that many species known to synthesize the polyamine spermidine lack the spermidine biosynthetic enzymes S-adenosylmethionine decarboxylase and spermidine synthase. We found that such species possess orthologues of the sym-norspermidine biosynthetic enzymes carboxynorspermidine dehydrogenase and carboxynorspermidine decarboxylase. By deleting these genes in the food-borne pathogen Campylobacter jejuni, we found that the carboxynorspermidine decarboxylase orthologue is responsible for synthesizing spermidine and not sym-norspermidine in vivo. In polyamine auxotrophic gene deletion strains of C. jejuni, growth is highly compromised but can be restored by exogenous sym-homospermidine and to a lesser extent by sym-norspermidine. The alternative spermidine biosynthetic pathway is present in many bacterial phyla and is the dominant spermidine route in the human gut, stomach, and oral microbiomes, and it appears to have supplanted the S-adenosylmethionine decarboxylase/spermidine synthase pathway in the gut microbiota. Approximately half of the gut Firmicutes species appear to be polyamine auxotrophs, but all encode the potABCD spermidine/putrescine transporter. Orthologues encoding carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase are found clustered with an array of diverse putrescine biosynthetic genes in different bacterial genomes, consistent with a role in spermidine, rather than sym-norspermidine biosynthesis. Due to the pervasiveness of ε-proteobacteria in deep sea hydrothermal vents and to the ubiquity of the alternative spermidine biosynthetic pathway in that phylum, the carboxyspermidine route is also dominant in deep sea hydrothermal vents. The carboxyspermidine pathway for polyamine biosynthesis is found in diverse human pathogens, and this alternative spermidine biosynthetic route presents an attractive target for developing novel antimicrobial compounds.     

Hepatocellular apoptosis in mice is associated with early upregulation of mitochondrial glucose metabolism

Hepatocyte death due to apoptosis is a hallmark of almost every liver disease. Manipulation of cell death regulatory steps during the apoptotic process is therefore an obvious goal of biomedical research. To clarify whether metabolic changes occur prior to the characteristic apoptotic events, we used ex vivo multinuclear NMR-spectroscopy to study metabolic pathways of [U-¹³C]glucose in mouse liver during Fas-induced apoptosis. We addressed whether these changes could be associated with protection against apoptosis afforded by Epidermal Growth Factor (EGF). Our results show that serum alanine and aspartate aminotransferase levels, caspase-3 activity, BID cleavage and changes in cellular energy stores were not observed before 3 h following anti-Fas injection. However, as early as 45 min after anti-Fas treatment, we observed upregulation of carbon entry (i.e. flux) from glucose into the Krebs-cycle via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) (up to 139% and 123% of controls, respectively, P < 0.001). This was associated with increased glutathione synthesis. EGF treatment significantly attenuated Fas-induced apoptosis, liver injury and the late decrease in energy stores, as well as the early fluxes through PDH and PC which were comparable to untreated controls. Using ex vivo multinuclear NMR-spectroscopic analysis, we have shown that Fas receptor activation in mouse liver time-dependently affects specific metabolic pathways of glucose. These early upregulations in glucose metabolic pathways occur prior to any visible signs of apoptosis and may have the potential to contribute to the initiation of apoptosis by maintaining mitochondrial energy production and cellular glutathione stores.     

Expression of mitochondrial HMGCoA synthase and glutaminase in the colonic mucosa is modulated by bacterial species.

The expression of the colonic mitochondrial 3-hydroxy 3-methyl glutaryl CoA (mHMGCoA) synthase, a key control site of ketogenesis from butyrate, is lower in germ-free (GF) than in conventional (CV) rats. In contrast, the activity of glutaminase is higher. The objective of this study was to investigate whether the intestinal flora can affect gene expression through short chain fatty acid (SCFA) and butyrate production. GF rats were inoculated with a conventional flora (Ino-CV) or with a bacterial strain producing butyrate (Clostridium paraputrificum, Ino-Cp) or not (Bifidobacterium breve, Ino-Bb). In the Ino-CV rats, mHMGCoA synthase expression was restored to the CV values 2 days after the inoculation, i.e. concomitantly with SCFA production. In the Ino-Cp group, but not in the Ino-Bb group, mHMGCoA synthase and glutaminase were expressed at the level observed in the CV rats. These data suggest that the intestinal flora, through butyrate production, could control the expression of colonic mHMGCoA synthase and glutaminase. These modifications in gene expression by butyrate in vivo seem unrelated to a modification of histone acetylation.     

Static suppression of tomato bacterial wilt by bacterial coagulation using a new functional polymer that coagulates bacterial cells and is highly biodegradable

Tomato bacterial wilt by Ralstonia solanacearum was suppressed by coagulation of bacterial cells without disinfection using a copolymer of methyl methacrylate with N-benzyl-4-vinylpyridinium chloride in a molar ratio of 3:1 (PMMA-co-BVP) as a polymeric coagulant for bacterial cells. When 10 mg/kg of PMMA-co-BVP was added to soil before transplanting of tomato seedlings, and 2 mg/kg was supplemented once a week after transplanting, a 51% reduction of appearance and a 54% reduction of index of symptoms were observed. PMMA-co-BVP did not exhibit bactericidal activity against R. solanacearum, and coagulation of the bacterial cells appeared to reduce the opportunity for infectious contact of roots of tomato with cells of R. solanacearum, and resulted in disease suppression. PMMA-co-BVP was shown to be highly biodegradable, and the half-life was 5.1 d when treated with activated sludge in soil.