Lung tissue extracellular matrix-derived hydrogels protect against radiation-induced lung injury by suppressing epithelial–mesenchymal transition

This study aimed to examine whether lung tissue extracellular matrix (ECM) hydrogels have protective effects on radiation-induced lung injury (RILI). The cytocompatibility and histocompatibility were tested for the obtained ECM-derived hydrogel. Sprague–Dawley rats were randomly divided into three groups (n = 18): control group (control); rats receiving irradiation and intratracheal injection of normal saline (IR + NS); and rats receiving irradiation and intratracheal injection of lung ECM-derived hydrogel (IR + ECM). The wet/dry weight ratio was used to evaluate the congestion and edema of the lungs. Histopathological analysis of lung tissues was performed using hemotoxylin and eosin staining and Masson's trichrome staining. Immunohistochemical staining and western blot analyses were carried out to determine the expression of epithelial–mesenchymal transition (EMT)-related proteins in lung tissues (E-cadherin, α-smooth muscle actin [α-SMA], and vimentin). In addition, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6), hydroxyproline, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were also evaluated. The ECM-derived hydrogels had good cytocompatibility and histocompatibility. ECM-derived hydrogel treatment improved lung histopathology injury and pulmonary edema. Higher expression of E-cadherin and lower expression of vimentin and α-SMA were found in the IR + ECM group compared with those in the IR + NS group. Hydroxyproline levels were reduced by ECM-derived hydrogel treatment compared with those in the IR + NS group. Obvious increases of TNF-α, IL-6, and TGF-β1 were identified following irradiation. Marked reductions in MDA content and increases in SOD were induced by ECM-derived hydrogel treatment in rats after radiation. ECM-derived hydrogels were shown to protect against RILI, potentially by reducing EMT, inflammation, and oxidative damage.     

Lamp-1 is upregulated in human glioblastoma cell lines induced to undergo apoptosis

Lysosome-associated membrane protein (LAMP)-1, one of the major protein components of the lysosomal membrane, is upregulated in the human glioblastoma cell lines, U-373 MG and LN-Z308, which undergo cisplatin-induced apoptosis. These human brain tumor cell lines demonstrated apoptosis in response to cisplatin/nifedipine treatment. Both cell lines demonstrated an apoptotic response by more than one criterion. Apoptosis was demonstrated by DNA fragmentation techniques such as DNA laddering, ApopTag in situ labeling, and an ELISA-based method of detecting liberated oligosomes. These cells also had characteristic morphologic changes and upregulation of bax consistent with apoptosis. LAMP-1 expression at the protein and mRNA level was examined and found to increase with cisplatin/nifedipine treatment. LAMP-1 expression was examined using indirect immunofluorescent staining, Northern blot analysis and Western blot analysis. The finding of an augmentation of LAMP-1 in these cells induced to die is enigmatic. These findings raise the possibility of LAMP-1 involvement in the apoptotic process.     

p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells

This study examined the effects of p53 gene status on DNA damage-induced cell death and chemosensitivity to various chemotherapeutic agents in non-small cell lung cancer (NSCLC) cells. A mutant p53 gene was introduced into cells carrying the wild-type p53 gene and also vice versa to introduce the wild-type p53 gene into cells carrying the mutant p53 gene. Chemosensitivity and DNA damage-induced apoptosis in these cells were then examined. This study included five cell lines, NCI-H1437, NCI-H727, NCI-H441 and NCI-H1299 which carry a mutant p53 gene and NCI-H460 which carries a wild-type p53 gene. Mutant p53-carrying cells were transfected with the wild-type p53 gene, while mutant p53 genes were introduced into NCI-H460 cells. These p53 genes were individually mutated at amino acid residues 143, 175, 248 and 273. The representative cell line NCI-H1437 cells transfected with wild-type p53 gene (H1437/wtp53) showed a dramatic increase in susceptibility to three anticancer agents (7-fold to cisplatin, 21-fold to etoposide, and 20-fold to camptothecin) compared to untransfected or neotransfected H1437 cells. An increase in chemosensitivity was also observed in wild-type p53 transfectants of H727, H441, H1299 cells. The results of chemosensitivity were consistent with the observations on apoptotic cell death. H1437/wtp53 cells, but not H1437 parental cells, exhibited a characteristic feature of apoptotic cell death that generated oligonucleosomal-sized DNA fragments. In contrast, loss of chemosensitivity and lack of p53-mediated DNA degradation in response to anticancer agents were observed in H460 cells transfected with mutant p53. These observations suggest that the increase in chemosensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In addition, our results also suggest that p53 gene status modulates the extent of chemosensitivity and the induction of apoptosis by different anticancer agents in NSCLC cells.     

Commentary: When does understanding phenotypic evolution require identification of the underlying genes?

Adaptive evolution is fundamentally a genetic process. Over the past three decades, characterizing the genes underlying adaptive phenotypic change has revealed many important aspects of evolutionary change. At the same time, natural selection is often fundamentally an ecological process that can often be studied without identifying the genes underlying the variation on which it acts. This duality has given rise to disagreement about whether, and under what circumstances, it is necessary to identify specific genes associated with phenotypic change. This issue is of practical concern, especially for researchers who study nonmodel organisms, because of the often enormous cost and labor required to “go for the genes.” We here consider a number of situations and questions commonly addressed by researchers. Our conclusion is that although gene identification can be crucial for answering some questions, there are others for which definitive answers can be obtained without finding underlying genes. It should thus not be assumed that considerations of “empirical completeness” dictate that gene identification is always desirable.     

Targeted disruption of p53 attenuates doxorubicin-induced cardiac toxicity in mice

Use of the chemotherapeutic agent doxorubicin (Dox) is limited by dose-dependent cardiotoxic effects. The molecular mechanism underlying these toxicities are incompletely understood, but previous results have demonstrated that Dox induces p53 expression. Because p53 is an important regulator of the cell birth and death we hypothesized that targeted disruption of the p53 gene would attenuate Dox-induced cardiotoxicity. To test this, female 6–8 wk old C57BL wild-type (WT) or p53 knockout (p53 KO) mice were randomized to either saline or Dox 20 mg/kg via intraperitoneal injection. Animals were serially imaged with high-frequency (14 MHz) two-dimensional echocardiography. Measurements of left ventricle (LV) systolic function as assessed by fractional shortening (FS) demonstrated a decline in WT mice as early as 4 days after Dox injection and by 2 wk demonstrated a reduction of 31± 16% (P < 0.05) from the baseline. In contrast, in p53 KO mice, LV FS was unchanged over the 2 wk period following Dox injection. Apoptosis of cardiac myocytes as measured by the TUNEL and ligase reactions were significantly increased at 24 h after Dox treatment in WT mice but not in p53 KO mice. After Dox injection, levels of myocardial glutathione and Cu/Zn superoxide dismutase were preserved in p53 KO mice, but not in WT animals. These observations suggest that p53 mediated signals are likely to play a significant role in Dox-induced cardiac toxicity and that they may modulate Dox-induced oxidative stress.These two authors equally contributed to this study.     

The Evolution of Control and Distribution of Adaptive Mutations in a Metabolic Pathway

In an attempt to understand whether it should be expected that some genes tend to be used disproportionately often by natural selection, we investigated two related phenomena: the evolution of flux control among enzymes in a metabolic pathway and properties of adaptive substitutions in pathway enzymes. These two phenomena are related by the principle that adaptive substitutions should occur more frequently in enzymes with greater flux control. Predicting which enzymes will be preferentially involved in adaptive evolution thus requires an evolutionary theory of flux control. We investigated the evolution of enzyme control in metabolic pathways with two models of enzyme kinetics: metabolic control theory (MCT) and Michaelis–Menten saturation kinetics (SK). Our models generate two main predictions for pathways in which reactions are moderately to highly irreversible: (1) flux control will evolve to be highly unequal among enzymes in a pathway and (2) upstream enzymes evolve a greater control coefficient then those downstream. This results in upstream enzymes fixing the majority of beneficial mutations during adaptive evolution. Once the population has reached high fitness, the trend is reversed, with the majority of neutral/slightly deleterious mutations occurring in downstream enzymes. These patterns are the result of three factors (the first of these is unique to the MCT simulations while the other two seem to be general properties of the metabolic pathways): (1) the majority of randomly selected, starting combinations of enzyme kinetic rates generate pathways that possess greater control for the upstream enzymes compared to downstream enzymes; (2) selection against large pools of intermediate substrates tends to prevent majority control by downstream enzymes; and (3) equivalent mutations in enzyme kinetic rates have the greatest effect on flux for enzymes with high levels of flux control, and these enzymes will accumulate adaptive substitutions, strengthening their control. Prediction 1 is well supported by available data on control coefficients. Data for evaluating prediction 2 are sparse but not inconsistent with this prediction.THEORETICAL research on the process of adaptation has focused primarily on describing the size and number of genetic changes underlying phenotypic change (Fisher 1930; Kimura 1983; Orr 1998, 2002, 2003). By contrast, comparatively little theoretical attention has been given to the question of whether certain genes or types of genes are preferentially involved in the process of adaptation. Yet the current debate over the relative importance of regulatory vs. structural genes in morphological evolution (Hoekstra and Coyne 2007; Stern and Orgogozo 2008) clearly indicates that this question is of interest to evolutionary biologists.One situation in which this question is pertinent is the evolution of characters that are influenced by the concentration of end products of metabolic pathways. Often change in end-product concentration can be achieved by substitutions in any one of several genes in the pathway. One example is the intensity of floral pigmentation. To a first approximation, final pigment concentration, and hence color intensity, can be viewed as being determined by the flux rate down the pigment biosynthetic pathway for a fixed time corresponding to the duration of floral development. More generally, any situation in which flux rate determines phenotype is likely to fall in this category. In such situations, metabolic control theory (MCT) (Kacser and Burns 1973) and similar approaches (Heinrich and Rapoport 1974; Savageau 1976) indicate that changes in flux can be achieved by changing the activity of any enzyme in the pathway. We seek to determine whether and, if so, why enzymes differ in the probability that they contribute to evolutionary change in pathway flux.It has been suggested as a general principle that enzymes with the greatest control over flux will be disproportionately involved in such evolutionary change (Hartl et al. 1985; Eanes 1999; Watt and Dean 2000). This argument is based on the theoretical expectation that the probability of fixation of an advantageous allele is roughly proportional to its selection coefficient (Hedrick 2000). Since mutations equivalent in terms of enzyme kinetic properties will have greater effects on flux, and hence on fitness, in enzymes with greater metabolic control, mutations in those enzymes will be substituted preferentially.While this argument is likely sound, it simply pushes back the question of which genes evolve preferentially to the question of which enzymes are expected to have greatest control over flux. Although we are unaware of any theoretical attempts to model the evolution of flux control, many authors have speculated about where in pathways control is expected to be highest.Kacser and Burns (1973) hypothesized that the magnitudes of flux control exerted by different enzymes may be very similar. This hypothesis was based on the result from MCT that in linear pathways, overall flux control can be shared by all enzymes. Since metabolic pathways often consist of many enzymes, each would be expected to have only a limited potential to influence flux. Subsequent theoretical analysis of this hypothesis demonstrated that a given flux is consistent with many different flux-control distributions, including, at one extreme, equal flux control by all enzymes and, at the other extreme, major control by one or a few enzymes and little control for all others (Mazat et al. 1996). However, the question of which of these possibilities, if any, are likely to be favored by selection has not been addressed.Another hypothesis, the epistatic or synergistic principle, predicts that control will be vested in a single enzyme at any given time, but will shift over time among enzymes (Dykhuizen et al. 1987; Keightley 1989; Bost et al. 2001) According to this hypothesis, starting from equal control among enzymes in the pathway, selection to increase (or decrease) flux will cause the activity of one enzyme to increase (decrease). This change results in a decrease (increase) in flux control for the enzyme that changes and an increase (decrease) in control for the other pathway enzymes, causing control to be unequally shared. While this argument seems plausible, there has been no analysis of whether over time all enzymes have an equal chance of having elevated control.Finally, Eanes'' (1999) review of enzyme polymorphisms found that control is often centered in enzymes at pathway branch points, which constitute the most upstream enzymes of their specific branch. Flowers et al. (2007) also demonstrated that branching enzymes tend to exhibit more adaptive substitutions than downstream enzymes as would be expected under the principle that evolutionary change will be concentrated in enzymes with the largest control coefficients. In addition, evolutionary changes in these enzymes may be favored because they allow organisms to modify flux allocation to alternate functions and track environmental fluctuations. This suggestion is supported by the “branch point effect,” a theoretical demonstration that control coefficients can dramatically shift between enzymes depending on the kinetic rates of the two competing enzymes (LaPorte et al. 1984). However, this study does not address the question of how the distribution of control is likely to evolve, but describes only which distributions of control are mathematically possible. Thus, Eanes (1999, p. 318) concludes his review, stating “all enzymes in [a] contributing pathway may not be equal; determining the rule[s] for these inequalities should be a major goal in studies of enzyme polymorphism.”A control coefficient (CC) indicates the degree to which flux through a pathway is altered by a small change in the activity of an enzyme (see appendix; this is equivalent to the sensitivity coefficient of Kacser and Burns 1973). The “rules” governing the distribution of control coefficients are determined by the biological evolution of metabolic systems. While research demonstrates that there are many possible distributions of control coefficients, none has examined which of these is most likely to evolve. The optimization of metabolic systems has been explored in detail (Heinrich et al. 1991, 1997; Heinrich and Schuster 1998). In these studies, however, the investigators employ as optimization criteria maximizing flux, maximizing transient times, or minimizing metabolic intermediates, criteria whose biological and evolutionary relevance is unclear.In an effort to understand how control is expected to be shared among enzymes and predict which enzymes are most likely to contribute to adaptive genetic changes, we present two models of the evolution of flux control in a simple linear pathway. The first model employs the framework of MCT. Although there have been many challenges to the MCT framework (Savageau 1976, 1992; Cornish-Bowden 1989; Savageau and Sorribas 1989), it should be made clear that our aim is not to construct a precisely parameterized model of a particular biological system, but to use this generalized framework to address a single, critically ignored question: What are the rules governing how control will evolve to be distributed among enzymes? The use of the MCT framework to address questions in evolutionary genetics is firmly established, with investigation focused on the molecular basis of dominance (Kacser and Burns 1981; Keightley 1996a; Phadnis and Fry 2005; but see Bagheri and Wagner 2004), the relationship between metabolic flux and fitness (Dykhuizen et al. 1987; Szathmary 1993), the amount of additive and nonadditive genetic variance in metabolic systems (Keightley 1989), whether this variation can be explained by mutation–selection balance (Clark 1991), and patterns of response of quantitative traits to selection (Keightley 1996b). The second model we examine, saturation kinetics (SK), is based on Michealis–Menten kinetics and enables us to relax one major assumption of MCT: that enzymes are far from saturation.Here we limit our analysis to linear pathways as an initial attempt to examine these issues. We find that for such pathways control coefficients will generally evolve to be unequal; that the magnitude of this inequality depends on the thermodynamic properties, rather than the kinetic properties, of each reaction step; that upstream enzymes tend to evolve higher control coefficients than downstream enzymes; and that upstream enzymes fix advantageous mutations in greater numbers, and those mutations have larger effects than in downstream enzymes.     

Ameliorating role of caffeic acid phenethyl ester (CAPE) against isoniazid-induced oxidative damage in red blood cells

Isoniazid (INH) still remains a first-line drug both for treatment and prophylaxis of tuberculosis, but various organs toxicity frequently develops in patients receiving this drug. We aimed to investigate possible toxic effects of INH on rat red blood cells (RBCs), and to elucidate whether Caffeic acid phenethyl ester (CAPE) prevents a possible toxic effect of INH. Experimental groups were designed as follows: control group, INH group, INH + CAPE group. Compared with the control, the INH caused a significant increase in superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels, and a decrease in glutathione peroxidase (GSH-Px) and catalase (CAT), which are recently used to monitor the development and extent of damage due to oxidative stresses. CAPE administration to INH group ameliorated above changes due to INH.