Role for CysJ Flavin Reductase in Molybdenum Cofactor-Dependent Resistance of Escherichia coli to 6-N-Hydroxylaminopurine

We have previously described a novel Escherichia coli detoxification system for the removal of toxic and mutagenic N-hydroxylated nucleobases and related compounds that requires the molybdenum cofactor. Two subpathways (ycbX and yiiM) were identified, each employing a novel molybdo activity capable of inactivating N-hydroxylated compounds by reduction to the corresponding amine. In the present study, we identify the cysJ gene product as one additional component of this system. While the CysJ protein has been identified as the NADPH:flavin oxidoreductase component of the CysJI sulfite reductase complex (CysJ₈I₄), we show that the role of CysJ in base analog detoxification is unique and independent of CysI and sulfite reductase. We further show that CysJ functions as a specific partner of the YcbX molybdoenzyme. We postulate that the function of CysJ in this pathway is to provide, via its NADPH:flavin reductase activity, the reducing equivalents needed for the detoxification reaction at the YcbX molybdocenter. In support of the proposed interaction of the CysJ and YcbX proteins, we show that an apparent CysJ-YcbX “hybrid” protein from two Vibrio species is capable of compensating for a double cysJ ycbX defect in E. coli.Mutagenic base analogs are chemically modified nucleobases that can be incorporated in the cellular metabolism through purine or pyrimidine salvage pathways. Once converted to the deoxynucleoside triphosphate (dNTP) level, they may participate in DNA replication in an error-prone manner because of their ambivalent base-pairing capacity (11). Such synthetic base analogs are often used as a sensitive tool for studying DNA replication fidelity, DNA repair, or the metabolism of nucleic acid precursors. Mutagenic base analogs such as 8-oxoguanine or 3-methyladenine can also be formed in vivo as a consequence of normal cellular metabolism or produced by chemical and physical factors, such as alkylating agents or ionizing radiation.An important group of mutagenic and cytotoxic analogs are the N-hydroxylated nucleobases (or ribosides) such as 6-N-hydroxylaminopurine (HAP), 2-amino-HAP, or N⁴-hydroxycytidine (15). Specifically, HAP was found to be a very strong mutagen in bacteria and fungi, as well as mammalian cells (2, 20, 27). Some data have suggested that HAP may also be formed in vivo under oxidative stress (30) or as a by-product of certain purine salvage/interconversion pathways (5, 22).The genetic control of HAP-induced mutagenesis has been studied in some detail in the yeast Saccharomyces cerevisiae and in the bacterium Escherichia coli. In S. cerevisiae, resistance to HAP depends primarily on genes involved in adjusting and regulating the DNA or RNA precursor pools (HAM1 [ITP/XTPase], AAH1 [adenine aminohydrolase], and ADE genes involved in de novo AMP biosynthesis) (34).In E. coli, the major pathway that protects cells against HAP and related N-hydroxylated compounds is controlled by the moa, moe, and mog genes, which are required for biosynthesis of molybdenum cofactor (MoCo) (18, 19). MoCo is an essential cofactor for a varied group of oxidoreductases that are widely distributed from bacteria to humans. Chemically, MoCo is a pterin derivative (molybdopterin) that coordinates a molybdenum atom that serves as a catalytic redox center (for reviews, see references 23, 28, and 29). Based on catalytic details and sequence homology, molybdopterin-containing enzymes have been divided in four families: the xanthine oxidase family, the sulfite oxidase family, the dimethyl sulfoxide (DMSO) reductase family, and the aldehyde ferredoxin oxidoreductase family (14, 16). However, our previous studies on the MoCo-dependent resistance to HAP showed that none of the known or putative E. coli members of these families are responsible for the major HAP resistance mechanism (19). Instead, we discovered that HAP resistance is dependent on two newly described proteins, YcbX and YiiM, that are characterized by a so-called MOSC domain (molybdenum cofactor sulfurase C-terminal domain) (1, 17). This domain was first described as part of eukaryotic MoCo sulfurases (MOSs) (1), and it most likely represents a novel class of MoCo-binding domain, as indicated by studies on two mammalian MOSC-containing proteins (mARC1 and mARC2) discovered in mitochondria (12, 13).Our studies in E. coli showed that cell-free bacterial extracts were capable of converting HAP to adenine by an N-reductive reaction (17). Importantly, this conversion was entirely dependent on the presence of MoCo and the YcbX or YiiM proteins (17). Consequently, we suggested that this reduction of HAP to adenine forms the basis of the in vivo MoCo-dependent detoxification in E. coli (17). Interestingly, the mammalian MOSC-containing proteins mARC1 and mARC2 were shown to mediate the reduction of the N-hydroxylated prodrug benzamidoxime to its active amino form benzamidine (12, 13). Thus, the reduction of N-hydroxylated compounds may be a defining feature for the broadly distributed MOSC proteins (1).Our previous analyses also revealed that the E. coli ycbX and yiiM genes define two independent subpathways within the MoCo-dependent system (17). This is illustrated in the overall scheme shown in Fig. ​Fig.1.1. MoCo is synthesized in a series of steps from GTP by-products of the moa, moe, and mog operons. MoCo is then used as a cofactor for the YcbX and YiiM proteins, which reduce the N-hydroxylated compound to the corresponding amino form. The ycbX and yiiM pathways are genetically distinct as determined by epistasis experiments (17). They also differ by their substrate specificity patterns: YcbX protects most strongly against HAP, whereas YiiM has its largest effects toward hydroxylamine (NH₂OH) (17).Open in a separate windowFIG. 1.Genetic framework for the major molybdenum cofactor (MoCo)-dependent pathways of detoxification of N-hydroxylated base analogs in E. coli (17). moaA to mogA indicate the series of genes required for MoCo biosynthesis (19, 28), while ycbX and yiiM represent the two independent subpathways identified within the MoCo-dependent pathway (17). Specifically, ycbX and yiiM produce apoenzymes that react with MoCo to form the active YcbX and YiiM proteins. The diagram also indicates the differential specificity of the two subpathways toward the model N-hydroxylated compounds used in our studies: 6-N-hydroxylaminopurine (HAP), 2-amino-HAP (AHAP), and hydroxylamine (NH₂OH). For simplicity, the diagram does not distinguish between the MPT and MGD forms of MoCo (19). As shown elsewhere (19), YcbX and YiiM likely employ the MPT form. One additional, minor pathway for HAP detoxification dependent on biotin sulfoxide reductase (an MGD-requiring enzyme) is observable only in the double ycbX yiiM-deficient background and is likewise not shown here (see reference 17 for details).Prior to the establishment of this scheme of YcbX and YiiM as molybdoproteins, we had entertained certain alternative possibilities for the precise function of the ycbX and yiiM open reading frames (ORFs), including a possible role in MoCo sulfuration (which is a required modification of MoCo in certain molybdoenzymes, such as xanthine oxidase) (23, 29). This sulfuration model was ultimately eliminated (17), but certain experiments related to this hypothesis yielded interesting further clues regarding the detailed mechanisms of HAP resistance. These observations included an unexpected HAP-sensitive phenotype for cysJ mutants as well as a noted sensitization of wild-type strains to HAP by l-cysteine. In the present work, we describe these experiments and show the cysJ gene to be an essential component of the ycbX branch of HAP resistance. In a related mechanism, the observed sensitization of wild-type strains by l-cysteine results from the suppression, by l-cysteine, of the cys regulon. Overall, our experiments suggest that CysJ is a specific protein partner of YcbX and that CysJ mediates the N-reductive reaction through its NADPH:flavin oxidoreductase activity. This activity provides reducing equivalents to its partner YcbX, which ultimately performs the reduction of HAP to nontoxic adenine at its molybdocenter.     

Mesenchymal stem cells inhibit generation and function of both CD34+-derived and monocyte-derived dendritic cells

Mesenchymal stem cells (MSCs) are not only able to evade the immune system, but they have also been demonstrated to exert profound immunosuppressive properties on T cell proliferation. However, their effect on the initiators of the immune response, the dendritic cells (DCs), are relatively unknown. In the present study, the effects of human MSCs on the differentiation and function of both CD34+ -derived DCs and monocyte-derived DCs were investigated. The presence of MSCs during differentiation blocked the differentiation of CD14+CD1a- precursors into dermal/interstitial DCs, without affecting the generation of CD1a+ Langerhans cells. In line with these observations, MSCs also completely prevented the generation of immature DCs from monocytes. The inhibitory effect of MSCs on DC differentiation was dose dependent and resulted in both phenotypical and functional modifications, as demonstrated by a reduced expression of costimulatory molecules and hampered capacity to stimulate naive T cell proliferation. The inhibitory effect of MSCs was mediated via soluble factors. Taken together, these data demonstrate that MSCs, next to the antiproliferative effect on T cells, have a profound inhibitory effect on the generation and function of both CD34+ -derived and monocyte-derived DCs, indicating that MSCs are able to modulate immune responses at multiple levels.     

Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 (hisG428, rfa, ΔuvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 (uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 (recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 (recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 (mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage—resulting in an antimutagenic effect on spontaneous mutation.     

Phosphorus intensity determines short-term P uptake by pigeon pea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> L.) grown in soils with differing P buffering capacity

Phosphorus (P) uptake by plant roots depends on P intensity (I) and P quantity (Q) in the soil. The relative importance of Q and I on P uptake is unknown for soils with large P sorption capacities because of difficulties in determining trace levels of P in the soil solution. We applied a new isotope based method to detect low P concentrations (<20 μg P l⁻¹). The Q factor was determined by assessment of the isotopically exchangeable P in the soil (E-value) and the I factor was determined by measurement of the P concentration in the pore water. A pot trial was set up using four soils with similar labile P quantities but contrasting P buffering capacities. Soils were amended with KH₂PO₄ at various rates and pigeon pea (Cajanus cajan L.) was grown for 25 days. The P intensity ranged between 0.0008 and 50 mg P l⁻¹ and the P quantity ranged between 10 and 500 mg P kg⁻¹. Shoot dry matter (DM) yield and P uptake significantly increased with increasing P application rates in all soils. Shoot DM yield and P uptake, relative to the maximal yield or P uptake, were better correlated with the P concentration in the pore water (R ² = 0.83–0.90) than with the E-value (R ²=0.40–0.53). The observed P uptakes were strongly correlated to values simulated using a mechanistic rhizosphere model (NST 3.0). A sensitivity analysis reveals that the effect of P intensity on the short-term P uptake by pigeon pea exceeded the effect of P quantity both at low and high P levels. However, DM yield and P uptake at a given P intensity consistently increased with increasing P buffering capacity (PBC). The experimental data showed that the intensity yielding 80% of the maximal P uptake was 4 times larger in the soil with the smallest PBC compared to the soil with the largest PBC. This study confirms that short-term P uptake by legumes is principally controlled by the P intensity in the soil, but is to a large extent also affected by the PBC of the soil. Section Editor: N. J. Barrow     

A dedicated Wnt secretion factor

The Wnt family of signaling proteins mediates cell-cell communication during development. In this issue of Cell, B?nziger et al. (2006) and Bartscherer et al. (2006) identify Wntless/Evi, a multipass transmembrane protein in the secretory pathway of Wnt-producing cells that promotes Wnt secretion.     

Kinetic analysis of bifidobacterial metabolism reveals a minor role for succinic acid in the regeneration of NAD+ through its growth-associated production

Several strains belonging to the genus Bifidobacterium were tested to determine their abilities to produce succinic acid. Bifidobacterium longum strain BB536 and Bifidobacterium animalis subsp. lactis strain Bb 12 were kinetically analyzed in detail using in vitro fermentations to obtain more insight into the metabolism and production of succinic acid by bifidobacteria. Changes in end product formation in strains of Bifidobacterium could be related to the specific rate of sugar consumption. When the specific sugar consumption rate increased, relatively more lactic acid and less acetic acid, formic acid, and ethanol were produced, and vice versa. All Bifidobacterium strains tested produced small amounts of succinic acid; the concentrations were not more than a few millimolar. Succinic acid production was found to be associated with growth and stopped when the energy source was depleted. The production of succinic acid contributed to regeneration of a small part of the NAD+, in addition to the regeneration through the production of lactic acid and ethanol.     

Heat-shock protein 27 is a major methylglyoxal-modified protein in endothelial cells

In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d-glucose produced approximately 2-fold higher levels of methylglyoxal but not 3-deoxyglucosone and glyoxal, as compared to 5 mM d-glucose. Under hyperglycaemic conditions, the methylglyoxal-arginine adduct argpyrimidine as detected with a specific antibody, but not N(e)-(carboxymethyl)lysine and N(e)-(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARgamma ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine-modified protein. With in-gel digestion and mass spectrometric analysis, we identified this major protein as heat-shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.     

Dissection of a metastatic gene expression signature into distinct components

Background

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.

Results

For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed.

Conclusion

Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.     

Subtotal nephrectomy plus coronary ligation leads to more pronounced damage in both organs than either nephrectomy or coronary ligation

Coexistence of chronic kidney disease (CKD) and heart failure (HF) in humans is associated with poor outcome. We hypothesized that preexistent CKD worsens cardiac outcome after myocardial infarction, and conversely that ensuing HF worsens progression of CKD. Subtotally nephrectomized (SNX) or sham-operated (CON) rats were subjected to coronary ligation (CL) or sham surgery in week 9 to realize four groups: CON, SNX, CON + CL, and SNX + CL. Blood pressure and renal function were measured in weeks 8, 11, 13, and 15. In week 16, cardiac hemodynamics and end-organ damage were assessed. Blood pressure was significantly lower in SNX + CL vs. SNX. Despite this, glomerulosclerosis was more severe in SNX + CL vs. SNX. Two weeks after CL, SNX + CL had more cardiac dilatation compared with CON + CL (end-diastolic volume index: 0.28 ± 0.04 vs. 0.19 ± 0.03 ml/100 g body wt; mean ± SD, P < 0.001), although infarct size was similar. During follow-up in SNX + CL, ejection fraction declined. Mortality was only observed in SNX + CL (2 out of 9). In SNX + CL, end-diastolic pressure (18 ± 4 mmHg) and tau (29 ± 9 ms), the time constant of active relaxation, were significantly higher compared with SNX (13 ± 3 mmHg, 20 ± 4 ms; P < 0.01) and CON + CL (11 ± 5 mmHg, 17 ± 2 ms; P < 0.01). The diameter of small arterioles in the myocardium was significantly decreased in SNX + CL vs. CON + CL (P < 0.01). Urinary excretion of NO metabolites was significantly lower in SNX + CL compared with both CL and SNX. This study demonstrates the existence of more heart and more kidney damage in a new model of combined CKD and HF than in the individual models. Such enhanced damage appears to be separate from systemic hemodynamic changes. Reduced nitric oxide availability may have played a role in both worsened glomerulosclerosis and cardiac diastolic function and appears to be a connector in the cardiorenal syndrome.     

Gene expression profiling of pituitary melanotrope cells during their physiological activation

The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the production of the pigment-dispersing peptide α-melanophore-stimulating hormone, which allows the animal to adapt its skin color to its environment. During adaptation to a dark background the melanotrope cells undergo remarkable changes characterized by dramatic increases in cell size and secretory activity. In this study we performed microarray mRNA expression profiling to identify genes important to melanotrope activation and growth. We show a strong increase in the expression of the immediate early gene (IEG) c-Fos and of the brain-derived neurotrophic factor gene (BDNF). Furthermore, we demonstrate the involvement of another IEG in the adaptation process, Nur77, and conclude from in vitro experiments that the expression of both c-Fos and Nur77 are partially regulated by the adenylyl cyclase system and calcium ions. In addition, we found a steady up-regulation of Ras-like product during the adaptation process, possibly evoked by BDNF/TrkB signaling. Finally, the gene encoding the 105-kDa heat shock protein HSPh1 was transiently up-regulated in the course of black-background adaptation and a gene product homologous to ferritin (ferritin-like product) was >100-fold up-regulated in fully black-adapted animals. We suggest that these latter two genes are induced in response to cellular stress and that they may be involved in changing the mode of mRNA translation required to meet the increased demand for de novo protein synthesis. Together, our results show that microarray analysis is a valuable approach to identify the genes responsible for generating coordinated responses in physiologically activated cells.