Effect of vitamin A treatment on superoxide dismutase-deficient yeast strains

Vitamin A (Vit A) is widely suggested to be protective against oxidative stress. However, different studies have been demonstrated the pro-oxidant effects of retinoids in several experimental models. In this work, we used the yeast Saccharomyces cerevisiae as a model organism to study the Vit A effects on superoxide dismutase (SOD)-deficient yeast strains. We report here that Vit A (10, 20 and 40 mg/ml) decreases the survival of exponentially growing yeast cells, especially in strains deficient in CuZnSOD (sod1Δ) and CuZnSOD/MnSOD (sod1Δsod2Δ). We also observed the protective effect of vitamin E against the Vit A-induced toxicity. Possible adaptation effects induced by sub-lethal oxidative stress were monitored by pre-, co- and post-treatment with the oxidative agent paraquat. The enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx), and the total glutathione content were determined after Vit A treatment. Our results showed that CuZnSOD represents an important defence against Vit A-generated oxidative damage. In SOD-deficient strains, the main defence against Vit A-produced reactive oxygen species (ROS) is GPx. However, the induction of GPx activity is not sufficient to prevent the Vit A-induced cell death in these mutants in exponential phase growth.     

Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP,LacY, ProW,AqpZ, MscS,and MscL

Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).Modern developments in fluorescence microscopy have led to a new understanding of the organization of bacterial cells, particularly protein and lipid localization (21, 56). Analysis of the subcellular localization of diverse proteins and lipids has shown that they are not uniformly distributed. The phospholipid cardiolipin (CL) localizes at the poles and septal regions (36), and there is evidence for segregation of phosphatidylethanolamine (PE) from phosphatidylglycerol (PG) in the membranes of living Escherichia coli cells (69). Localization of many proteins that are integral or peripheral to the cytoplasmic membrane has been studied by fusing them to green fluorescent protein (GFP) (or its derivatives), and it is possible to classify the fusion proteins according to their subcellular localization. The first group, comprised of proteins that are concentrated at the cell poles, includes chemoreceptors (31, 62), the lactose permease LacY (43), and the metabolic sensor kinases DcuS and CitA (55). Members of the second group form helices that extend from pole to pole and include MreB (25), MinD (57), the Sec protein export system (58), and RNase E, which is the main component of the RNA degradosome in E. coli (67). Other proteins may appear to be similarly distributed due to their association with the Sec system (58). Members of the third group are uniformly distributed and include the mechanosensitive channel MscL (45) and the sensor kinase KdpD (32).The polar localization of proteins appears to be a critical feature of the complicated internal localization of bacteria. For example, it is important for temporally and spatially accurate placement of the septum during cell division (15). However, the mechanism of protein organization at bacterial cell poles is still unclear, and in many cases its functional role has not been determined. Do the poles merely serve as a receptacle for proteins, superstructures, or membrane domains with no functional effects, or is this location functionally important for membrane proteins and lipids?Recent evidence indicates that the subcellular localization of the transporter ProP in E. coli is related to membrane phospholipid composition, cardiolipin localization, and ProP function (51, 52). E. coli cells from cultures grown to exponential phase contain mostly the zwitterionic phospholipid PE (approximately 75 mol%) and the anionic phospholipids PG (approximately 20 mol%) and CL (approximately 5 mol%) (8). (Note that cardiolipin is diphosphatidylglycerol.) However, the phospholipid composition depends on the bacterial growth conditions. We found that the proportion of CL among E. coli lipids varies directly with growth medium osmolality (68), and increased CL synthesis was at least partially attributed to regulation of the cls locus encoding cardiolipin synthase (52). There is residual CL in cls bacteria, indicating that there is an alternative pathway for CL synthesis (51). The CL-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to show that CL clusters at the poles and septa in growing E. coli cells (36, 52). This result was corroborated by analyzing the phospholipid composition of E. coli minicells (DNA-free cells resulting from asymmetric cell division) (24, 51).ProP is an osmosensory transporter that senses increasing osmolality and responds by mediating the cytoplasmic accumulation of organic osmolytes (e.g., proline, glycine betaine, and ectoine). Biochemical regulation of the ProP protein ensures that ProP activity increases with increasing assay medium osmolality (49). We showed that ProP and CL colocalize at the poles and near the septa of dividing E. coli cells and that the polar concentration of ProP correlates with the polar concentration of CL (52). Moreover, we showed that the osmolality required to activate ProP increased in parallel to the CL content when E. coli was cultivated in media with increasing osmolality (51, 52, 68). The osmolality required to activate ProP was also a direct function of CL content in proteoliposomes reconstituted with purified ProP (51). We concluded that concentration at the cell poles controlled the osmoregulatory function of ProP by placing the transporter in a cardiolipin-rich environment.To determine whether CL-dependent membrane protein localization is a general phenomenon in E. coli, we compared the subcellular localization of ProP with that of its paralogue LacY, a well-characterized lactose transporter (16). LacY and ProP are both members of the major facilitator superfamily and H⁺ symporters. LacY transports the nutrient lactose, and LacY activity decreases while ProP activity increases with increasing osmolality (9). Nagamori et al. reported polar localization of a LacY-GFP fusion protein in E. coli (43). We confirmed this observation and demonstrated that, in contrast to the behavior of ProP, the polar concentration of LacY did not correlate with the polar concentration of CL (51).In this work we further explored the relationship between CL and protein localization in E. coli. We compared ProP with other proteins related to cellular osmoregulation. Bacteria use arrays of osmoregulatory mechanisms to survive and function when the osmotic pressure of their environment changes. In E. coli, the aquaporin AqpZ mediates transmembrane water flux, the transporters ProP, ProU, BetT, and BetU mediate organic osmolyte accumulation at high osmotic pressure, and the mechanosensitive (MS) channels MscL and MscS mediate solute efflux in response to osmotic downshock (71). Localization of these proteins might be expected since AqpZ might influence cell morphology changes by accelerating water flux at particular positions on the cell surface and the pressure sensitivities of MscL and MscS are known to depend on membrane curvature in vitro (18).For ProP and LacY, we labeled the inserted peptide tag CCPGCC with the biarsenical fluorescein reagent FlAsH-EDT₂ (fluorescein arsenical helix binder, bis-EDT adduct) (1, 2) to examine the subcellular localization of AqpZ, the integral membrane component ProW of the osmoregulatory ATP-binding cassette (ABC) transporter ProU, and the MS channel proteins MscS and MscL in cls⁺ and cls bacteria. Fluorescence microscopy was used to determine the proportion of cells with labeled protein concentrated at the poles as a function of bacterial CL content and protein expression level. For ProP, the frequency with which MscS was concentrated at cell poles was proportional to the level and polar concentration of CL. LacY concentrated at the cell poles at a high and CL-independent frequency. The frequencies with which AqpZ, MscL, and ProW concentrated at the cell poles and septa were low (up to 12%) and CL independent.     

Context-dependent cell cycle checkpoint abrogation by a novel kinase inhibitor

Background

Checkpoint kinase 1 and 2 (Chk1/Chk2), and the Aurora kinases play a critical role in the activation of the DNA damage response and mitotic spindle checkpoints. We have identified a novel inhibitor of these kinases and utilized this molecule to probe the functional interplay between these two checkpoints.

Principal Findings

Fragment screening, structure guided design, and kinase cross screening resulted in the identification of a novel, potent small molecule kinase inhibitor (VER-150548) of Chk1 and Chk2 kinases with IC₅₀s of 35 and 34 nM as well as the Aurora A and Aurora B kinases with IC₅₀s of 101 and 38 nM. The structural rationale for this kinase specificity could be clearly elucidated through the X-ray crystal structure. In human carcinoma cells, VER-150548 induced reduplication and the accumulation of cells with >4N DNA content, inhibited histone H3 phosphorylation and ultimately gave way to cell death after 120 hour exposure; a phenotype consistent with cellular Aurora inhibition. In the presence of DNA damage induced by cytotoxic chemotherapeutic drugs, VER-150548 abrogated DNA damage induced cell cycle checkpoints. Abrogation of these checkpoints correlated with increased DNA damage and rapid cell death in p53 defective HT29 cells. In the presence of DNA damage, reduplication could not be observed. These observations are consistent with the Chk1 and Chk2 inhibitory activity of this molecule.

Conclusions

In the presence of DNA damage, we suggest that VER-150548 abrogates the DNA damage induced checkpoints forcing cells to undergo a lethal mitosis. The timing of this premature cell death induced by Chk1 inhibition negates Aurora inhibition thereby preventing re-entry into the cell cycle and subsequent DNA reduplication. This novel kinase inhibitor therefore serves as a useful chemical probe to further understand the temporal relationship between cell cycle checkpoint pathways, chemotherapeutic agent induced DNA damage and cell death.     

Responses of human endothelial cells to pathogenic and non-pathogenic Leptospira species

Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis.     

The canonical Wnt pathway in embryonic axis polarity

The canonical Wnt pathway plays crucial roles in multiple developmental processes, including in axis specification. Throughout the animal kingdom, this pathway has been reported to drive patterning of axes as different as the animal-vegetal axis in echinoderms to the dorsal-ventral axis in vertebrates. Intriguingly enough, this pathway appears structurally and functionally well conserved during evolution. However, differences between these phyla are observed that explain how a same pathway can mediate establishment of two such apparently distinct axes. This review compares the axis specification processes used in two evolutionarily distant embryos, the sea urchin and Xenopus.     

Cyclooxygenase-2 directly regulates gene expression of P450 Cyp19 aromatase promoter regions pII, pI.3 and pI.7 and estradiol production in human breast tumor cells

The present studies evaluated the direct effects of the presence of human cyclooxygenase-2 (Cox-2) on gene expression of specific promoter regions of the P450 Cyp19 enzyme aromatase enzyme and its product, estradiol, in Cox-2 null estrogen-dependent MCF-7 breast tumor cells and in a stable clone of MCF-7 cells containing transfected Cox-2 cDNA, designated as MCF-7/Cox-2 Clone 10. Clone 10 human breast tumor cells have significantly increased gene expression of total mRNA of the P450 Cyp19 enzyme aromatase, with high levels of gene expression of specific aromatase promoter (p) regions pII, pI.3, and p1.7, with no significant change in mRNA levels of p1.4. Clone 10 human breast tumor cells produced significantly increased amounts of both prostaglandin E2 (PGE2) derived from Cox-2 enzyme activity and estradiol derived from aromatase enzyme activity (p<0.01), compared to MCF-7/vector control cells. The greatest inhibition of PGE2 or estradiol production was observed by the combination of the selective Cox-2 inhibitor celecoxib (25 microM) and the aromatase inhibitor, formestane (10nM) (p<0.01). The greatest anti-proliferative effect in Cox-2 null MCF-7/vector control cells was observed with the combination of 25 microM celecoxib and 10nM formestane but not with 10 microM celecoxib, suggesting that there are Cox-2-independent mechanisms involved in the anti-proliferative effect of this agent at doses greater than 10 microM. Celecoxib (25 microM) also significantly inhibited proliferation of MCF-7/Cox-2 Clone 10 human breast tumor cells, with no further anti-proliferative activity with the addition of 10 nM formestane observed at either 24 or 48 h of treatment. These studies demonstrate that Cox-2 directly regulates gene expression of specific aromatase promoter regions and regulates aromatase enzyme activity. Agents that inhibit Cox-2 or block the biological effects of PGE2 may be useful in significantly limiting aromatase activity and proliferation of human breast tumor cells regardless of the presence of Cox-2. In addition, the unique human breast tumor cell model used in these studies may be a useful tool in identifying the spectrum of activities of agents that block the biological effects of PGE2 and estradiol.     

Abnormal cardiac energetics in patients carrying the A3243G mtDNA mutation measured in vivo using phosphorus MR spectroscopy

Cardiomyopathy is a frequent cause of morbidity and mortality in patients carrying the A3243G transition in the mitochondrial DNA (mtDNA) tRNALeu(UUR) gene, the most common heteroplasmic single mtDNA defect. We used phosphorus magnetic resonance spectroscopy (31P-MRS) to look for evidence of an in vivo bioenergetics defect in patients carrying the A3243G mtDNA mutation with and without echocardiographic signs of left ventricle hypertrophy (LVH). Eight patients, three with LVH, carrying the A3243G mtDNA mutation and 10 healthy subjects underwent one-dimensional chemical shift imaging 31P-MRS. In the patients, mean cardiac phosphocreatine to adenosine triphosphate ratio (PCr/ATP) (1.55 +/- 0.58) was significantly reduced compared to the control group (2.34 +/- 0.14; P < 0.001). Cardiac PCr/ATP was within the normal range only in one case that showed normal echocardiography. Our results point to a central role of bioenergetics deficit in the development of cardiac hypertrophy in patients with the A3243G mtDNA mutation. Impaired cardiac energy metabolism in patients with normal echocardiography suggests that the enhancement of mitochondrial function may be beneficial not only to patients with cardiac hypertrophy but also to those patients carrying the mutation in the absence of signs of cardiac hypertrophy and/or dysfunction but with cardiac bioenergetics deficit.     

Cloning and Targeted Disruption of MLG1, a Gene Encoding Two of Three Extracellular Mixed-Linked Glucanases of Cochliobolus carbonum

Mixed-linked glucanases (MLGases), which are extracellular enzymes able to hydrolyze β1,3-1,4-glucans (also known as mixed-linked glucans or cereal β-glucans), were identified in culture filtrates of the plant-pathogenic fungus Cochliobolus carbonum. Three peaks of MLGase activity, designated Mlg1a, Mlg1b, and Mlg2, were resolved by cation-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Mlg1a and Mlg1b also hydrolyze β1,3-glucan (laminarin), whereas Mlg2 does not degrade β1,3-glucan but does degrade β1,4-glucan to a slight extent. Mlg1a, Mlg1b, and Mlg2 have monomer molecular masses of 33.5, 31, and 29.5 kDa, respectively. The N-terminal amino acid sequences of Mlg1a and Mlg1b are identical (AAYNLI). Mlg1a is glycosylated, whereas Mlg1b is not. The gene encoding Mlg1b, MLG1, was isolated by using PCR primers based on amino acid sequences of Mlg1b. The product of MLG1 has no close similarity to any known protein but does contain a motif (EIDI) that occurs at the active site of MLGases from several prokaryotes. An internal fragment of MLG1 was used to create mlg1 mutants by transformation-mediated gene disruption. The total MLGase and β1,3-glucanase activities in culture filtrates of the mutants were reduced by approximately 50 and 40%, respectively. When analyzed by cation-exchange HPLC, the mutants were missing the two peaks of MLGase activity corresponding to Mlg1a and Mlg1b. Together, the data indicate that Mlg1a and Mlg1b are products of the same gene, MLG1. The growth of mlg1 mutants in culture medium supplemented with macerated maize cell walls or maize bran and the disease symptoms on maize were identical to the growth and disease symptoms of the wild type.     

Population Genetics of Drosophila Amylase. I. Genetic Control of Tissue-Specific Expression in D. PSEUDOOBSCURA

Drosophila pseudoobscura is polymorphic for tissue-specific expression of alpha-amylase in adult midguts. This enzyme is encoded by a single locus, Amy, on the third chromosome. In this paper we show: (1) Up to about 12 days post-eclosion, the midgut activity patterns remain stable; after 12 days areas not showing activity previously begin to show activity. Thus, the genes controlling the expression of Amy are temporally acting. (2) Diet affects the quantitative, but not the qualitative, expression of Amy. (3) The expression of Amy in adult midguts is under genetic control. Selection for different frequencies of patterns is possible; realized heritabilities are 0.20 to 0.50. Partial linkage with third chromosome inversions has been demonstrated; the genes or elements controlling Amy expression are not, however, confined to the third chromosome. (4) The genetic elements controlling tissue-specific expression of amylase do not coordinately control the expression of five other "digestive-type" enzymes that were studied.--This polymorphism appears to be analogous to that studied by Abraham and Doane (1978) in D. melanogaster, wherein they have mapped regulatory genes.