Subtractive Strategy Research Articles

Identification of up-regulated genes in tea leaves under mild infestation of green leafhopper

Transcriptional changes accompany induced resistance of plants against insect feeding, and monitoring transcriptional reorganization triggered in response to herbivory is an essential step for deciphering the molecular basis of the resistance. To isolate up-regulated genes in tea leaves under mild infestation of green leafhopper ( Empoasca vitis Göthe), a subtractive cDNA library was constructed using the suppression subtractive hybridization strategy. Subsequent differential screening, DNA sequencing analyses, and real-time PCR analyses identified 21 unique genes up-regulated by leafhopper feeding from the library. Among the genes likely to be directly involved in defense, those encoding pathogen-related protein and type 2 ribosome-inactivating protein were found in the subtractive library. In addition, genes involved in the CBL (calcineurin B-like)–CIPK (CBL-interacting protein kinases) pathway, cell wall fortification, secondary metabolite synthesis, water transport, and oxidative stress protection were identified in our experiment. Besides, a number of genes that have not been previously associated with defense responses against insects were isolated.

Helping Students to Connect Subtraction Strategies Improves Mathematical Reasoning for Students and Teachers

After administering an end of unit assessment written by the school’s math program, teachers of three second grade classes in a New York City school noticed a majority of the students had not demonstrated mastery of subtracting two, two-digit numbers. The teachers worked with the school’s math coach to implement an instructional unit that required students to make connections among three different subtraction strategies. Implementation of the unit resulted in improved mathematical reasoning for students and teachers in addition to an improvement in students’ subtraction skills.

Osteopontin enhances HIV replication and is increased in the brain and cerebrospinal fluid of HIV-infected individuals

Despite effective and widely available suppressive anti-HIV therapy, the prevalence of mild neurocognitive dysfunction continues to increase. HIV-associated neurocognitive disorder (HAND) is a multifactorial disease with sustained central nervous system inflammation and immune activation as prominent features. Inflammatory macrophages, HIV-infected and uninfected, play a central role in the development of HIV dementia. There is a critical need to identify biomarkers and to better understand the molecular mechanisms leading to cognitive dysfunction in HAND. In this regard, we identified through a subtractive hybridization strategy osteopontin (OPN, SPP1, gene) an inflammatory marker, as an upregulated gene in HIV-infected primary human monocyte-derived macrophages. Knockdown of OPN in primary macrophages resulted in a threefold decrease in HIV-1 replication. Ectopic expression of OPN in the TZM-bl cell line significantly enhanced HIV infectivity and replication. A significant increase in the degradation of the NF-κB inhibitor, IκBα and an increase in the nuclear-to-cytoplasmic ratio of NF-κB were found in HIV-infected cells expressing OPN compared to controls. Moreover, mutation of the NF-κB binding domain in the HIV-LTR abrogated enhanced promoter activity stimulated by OPN. Interestingly, compared to cerebrospinal fluid from normal and multiple sclerosis controls, OPN levels were significantly higher in HIV-infected individuals both with and without neurocognitive disorder. OPN levels were highest in HIV-infected individuals with moderate to severe cognitive impairment. Moreover, OPN was significantly elevated in brain tissue from HIV-infected individuals with cognitive disorder versus those without impairment. Collectively, these data suggest that OPN stimulates HIV-1 replication and that high levels of OPN are present in the CNS compartment of HIV-infected individuals, reflecting ongoing inflammatory processes at this site despite anti-HIV therapy.

Effect of Organ Enhancement and Habitus on Estimation of Unenhanced Attenuation at Contrast-Enhanced Dual-Energy MDCT: Concepts for Individualized and Organ-Specific Spectral Iodine Subtraction Strategies

The purpose of this study was to assess whether habitus and organ enhancement influence iodine subtraction and should be incorporated into spectral subtraction algorithms. This study included 171 patients. In the unenhanced phase, MDCT was performed with single-energy acquisition (120 kVp, 250 mAs) and in the parenchymal phase with dual-energy acquisitions (80 kVp, 499 mAs; 140 kVp, 126 mAs). Habitus was determined by measuring trunk diameters and calculating circumference. Iodine subtraction was performed with input parameters individualized to muscle, fat, and blood ratio. Attenuation of the liver, pancreas, spleen, kidneys, and aorta was assessed in truly and virtually unenhanced image series. Pearson analysis was performed to correlate habitus with the input parameters. Analysis of truly unenhanced and virtually unenhanced images was performed with the Student t test; magnitude of variation was evaluated with Bland-Altman plots. Correction strategies were derived from organ-specific regression analysis of scatterplots of truly unenhanced and virtually unenhanced attenuation and implemented in a pixel-by-pixel approach. Analysis of individual organ correction and truly unenhanced attenuation was performed with the Student t test. The correlations between habitus and blood ratio (r = 0.694) and attenuation variation of fat at 80 kVp (r = -0.468) and 140 kV (r = -0.454) were confirmed. Although overall mean attenuation differed by no more than 10 HU between truly and virtually unenhanced scans overall, these differences varied by organ and were large in individual patients. Paired comparisons of truly and virtually unenhanced measurements differed significantly for liver, spleen, pancreas, kidneys, and aortic blood pool (p < 0.001 for all comparisons), but paired comparisons of truly unenhanced and individually organ-corrected measurements did not differ when organ- and habitus-based correction strategies were applied (p > 0.38 for all comparisons). Habitus and organ enhancement influence virtually unenhanced imaging and should be incorporated into spectral subtraction algorithms.

Use of indirect addition in adults’ mental subtraction in the number domain up to 1,000

This study examined adults' use of indirect addition and direct subtraction strategies on multi-digit subtractions in the number domain up to 1,000. Seventy students who differed in their level of arithmetic ability solved multi-digit subtractions in one choice and two no-choice conditions. Against the background of recent findings in elementary subtraction, we manipulated the size of the subtrahend compared to the difference and only selected items with large distances between these two integers. Results revealed that adults frequently and efficiently apply indirect addition on multi-digit subtractions, yet adults with higher arithmetic ability performed more efficiently than those with lower arithmetic ability. In both groups, indirect addition was more efficient than direct subtraction both on subtractions with a subtrahend much larger than the difference (e.g., 713 - 695) and on subtractions with a subtrahend much smaller than the difference (e.g., 613 - 67). Unexpectedly, only adults with lower arithmetic ability fitted their strategy choices to their individual strategy performance skills. Results are interpreted in terms of mathematical and cognitive perspectives on strategy efficiency and adaptiveness.

Abnormal resting state corticolimbic blood flow in depressed unmedicated patients with major depression: A 15O‐H2O PET study

We investigated the differences in the resting state corticolimbic blood flow between 20 unmedicated depressed patients and 21 healthy comparisons. Resting state cerebral blood flow (CBF) was measured with H(2)(15)O PET. Anatomical MRI scans were performed on an Elscint 1.9 T Prestige system for PET-MRI coregistration. Significant changes in cerebral blood flow indicating neural activity were detected using an ROI-free image subtraction strategy. In addition, the resting blood flow in patients was correlated with the severity of depression as measured by HAM-D scores. Depressed patients showed decreases in blood flow in right anterior cingulate (Brodmann areas 24 and 32) and increased blood flow in left and right posterior cingulate (Brodmann areas 23, 29, 30), left parahippocampal gyrus (Brodmann area 36), and right caudate compared with healthy volunteers. The severity of depression was inversely correlated with the left middle and inferior frontal gyri (Brodmann areas 9 and 47) and right medial frontal gyrus (Brodmann area 10) and right anterior cingulate (Brodmann areas 24, 32) blood flow, and directly correlated with the right thalamus blood flow. These findings support previous reports of abnormalities in the resting state blood flow in the limbic-frontal structures in depressed patients compared to healthy volunteers.

Additive and subtractive resilience strategies as enablers of biographical reinvention: A qualitative study of ex-smokers and never-smokers

The notion of developing resilience is becoming increasingly important as a way of responding to the social determinants of poor health, particularly in disadvantaged groups. It is hypothesized that resilient individuals and communities are able to ‘bounce back’ from the adversities they face. This paper explores the processes involved in building resilience as an outcome in relation to both quitting smoking and never smoking. The study involved 93 qualitative, oral-history interviews with participants from population groups with high and enduring smoking rates in Adelaide, Australia, and was essentially interested in how some people in these groups managed to quit or never start smoking in the face of adversities, in comparison to a group of smokers. Our key findings relate to what we call additive and subtractive resilience strategies, which focus on the practices, roles and activities that individuals either ‘took on’ or ‘left behind’ in order to quit smoking or remain abstinent. The theoretical lenses we use to understand these resilience strategies relate to biographical reinforcement and biographical reinvention, which situate the resilience strategies in a broader ‘project of the self’, often in relation to attempting to develop ‘healthy bodies’ and ‘healthy biographies’.

A new dominant peroxiredoxin allele identified by whole-genome re-sequencing of random mutagenized yeast causes oxidant-resistance and premature aging

The combination of functional genomics with next generation sequencing facilitates new experimental strategies for addressing complex biological phenomena. Here, we report the identification of a gain-of-function allele of peroxiredoxin (thioredoxin peroxidase, Tsa1p) via whole-genome re-sequencing of a dominantSaccharomyces cerevisiae mutant obtained by chemical mutagenesis. Yeast strain K6001, a screening system for lifespan phenotypes, was treated with ethyl methanesulfonate (EMS). We isolated an oxidative stress-resistant mutant (B7) which transmitted this phenotype in a background-independent, monogenic and dominant way. By massive parallel pyrosequencing, we generated an 38.8 fold whole-genome coverage of the strains, which differed in 12,482 positions from the reference (S288c) genome. Via a subtraction strategy, we could narrow this number to 13 total and 4 missense nucleotide variations that were specific for the mutant. Via expression in wild type backgrounds, we show that one of these mutations, exchanging a residue in the peroxiredoxin Tsa1p, was responsible for the mutant phenotype causing background-independent dominant oxidative stress-resistance. These effects were not provoked by altered Tsa1p levels, nor could they be simulated by deletion, haploinsufficiency or over-expression of the wild-type allele. Furthermore, via both a mother-enrichment technique and a micromanipulation assay, we found a robust premature aging phenotype of this oxidant-resistant strain. Thus, TSA1-B7 encodes for a novel dominant form of peroxiredoxin, and establishes a new connection between oxidative stress and aging. In addition, this study shows that the re-sequencing of entire genomes is becoming a promising alternative for the identification of functional alleles in approaches of classic molecular genetics.

Quantitative Organization of GABAergic Synapses in the Molecular Layer of the Mouse Cerebellar Cortex

In the cerebellar cortex, interneurons of the molecular layer (stellate and basket cells) provide GABAergic input to Purkinje cells, as well as to each other and possibly to other interneurons. GABAergic inhibition in the molecular layer has mainly been investigated at the interneuron to Purkinje cell synapse. In this study, we used complementary subtractive strategies to quantitatively assess the ratio of GABAergic synapses on Purkinje cell dendrites versus those on interneurons. We generated a mouse model in which the GABAA receptor α1 subunit (GABAARα1) was selectively removed from Purkinje cells using the Cre/loxP system. Deletion of the α1 subunit resulted in a complete loss of GABAAR aggregates from Purkinje cells, allowing us to determine the density of GABAAR clusters in interneurons. In a complementary approach, we determined the density of GABA synapses impinging on Purkinje cells using α-dystroglycan as a specific marker of inhibitory postsynaptic sites. Combining these inverse approaches, we found that synapses received by interneurons represent approximately 40% of all GABAergic synapses in the molecular layer. Notably, this proportion was stable during postnatal development, indicating synchronized synaptogenesis. Based on the pure quantity of GABAergic synapses onto interneurons, we propose that mutual inhibition must play an important, yet largely neglected, computational role in the cerebellar cortex.

Direct detection of galaxy stellar halos: NGC 3957 as a test case

We present a direct detection of the stellar halo of the edge-on S0 galaxy NGC 3957, using ultra-deep VLT/VIMOS V and R images. This is achieved with a sky subtraction strategy based on infrared techniques. These observations allow us to reach unprecedented high signal-to-noise ratios up to 15 kpc away from the galaxy center, rendering photon-noise negligible. The 1 sigma detection limits are R = 30.6 mag/arcsec^2 and V = 31.4 mag/arcsec^2. We conduct a thorough analysis of the possible sources of systematic errors that could affect the data: flat-fielding, differences in CCD responses, scaling of the sky background, the extended halo itself, and PSF wings. We conclude that the V-R colour of the NGC 3957 halo, calculated between 5 and 8 kpc above the disc plane where the systematic errors are modest, is consistent with an old and preferentially metal-poor normal stellar population, like that revealed in nearby galaxy halos from studies of their resolved stellar content. We do not find support for the extremely red colours found in earlier studies of diffuse halo emission, which we suggest might have been due to residual systematic errors.

Dark fringe operation and noise reduction with optical subtraction approach and power allocation

We demonstrate a technique to achieve the reduction of excess noise with dark fringe operation by optical subtraction and power allocation strategy. By performing the subtraction in optical way, the excess noise resulted from the photocurrent is eliminated and the phase information can be extracted with single photoreceiver. Meanwhile, by allocating more optical power to the probe beam, the DC background and the signal amplitude of the photocurrent become very small simultaneously so that the dark fringe operation is achieved. We compare the spectral distributions resulted from the proposed technique with that from the widely employed balanced detection and the modified balanced detection techniques which both are based on electronic subtraction. It is found that the experimental results closely agree to the theoretical predictions.

A Novel Neutralizing Epitope of Human Cytomegalovirus Glycoprotein M Screened by Phage Display*

Human cytomegalovirus glycoprotein complex Ⅱ (gC Ⅱ ) consists of two glycoproteins, gM and gN. Although gC Ⅱ specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report on the generation of virus-neutralizing antibodies by immunizing with one epitope of gM. The epitope, termed MAD, was screened from random phage peptide library by subtractive strategy. The peptide sequence of MAD was highly homologous with 32～38 amino acids of HCMV gM. Mice immunized with MAD coupled with keyhole limpet hemocyanin (KLH) could produce specific antibodies against MAD, and the antibodies obtained could bind not only native HCMV particles, but also the recombinant gM30～78 peptide. ELISA analysis results showed that MAD could specifically bind HCMV-positive human serum samples. Virus-neutralizing assay results demonstrated that the antibodies against MAD could inhibit HCMV strain AD169 entering the human embryonic lung cells. The results suggested that MAD could be used as a new potential protective antigen in the development of HCMV vaccine.

Strategy instruction in Parkinson's disease: Influence on cognitive performance

ObjectivesThough strategic deficits are extensively investigated in Parkinson's disease (PD), little is known about the effects of instruction for PD patients. Thus, we compared the ability to internally generate a cognitive strategy with the ability to use a strategy after elaborate strategy instruction. MethodsPatients with PD (n=14) and matched healthy controls (n=22) were administered a Numerosity Judgement task in which they had to determine different numerosities of blocks presented in a square grid. In more complex task configurations, healthy participants tend to use a subtraction strategy. Participants in our study were confronted with a counting condition (A), a strategy initiation condition without instruction (B), and a strategy elaboration and strategy training condition (C). ResultsPatients and controls were comparable with respect to basic cognitive measures. PD patients and controls performed equivalently within the counting condition (A), but patients needed significantly more trials to initiate the subtraction strategy. With the exception of 1 PD patient, all patients were able to internally initiate the strategy (condition B). In condition C, both groups increased reaction times, but patients were significantly slower than controls. Moreover, only patients significantly increased error rates after strategy instruction. ConclusionAs long as sufficient time is provided for solving the task, results do not show a general deficit in the ability to internally generate a cognitive strategy in PD. Failures in strategy utilization strongly depend on cognitive load (working memory, executive functions). This bears important implications for the neuropsychological rehabilitation of PD patients.

Scanning the Cell Surface Proteome of Cancer Cells and Identification of Metastasis-Associated Proteins Using a Subtractive Immunization Strategy

Identification of the cell surface proteome and comparison of their expression between cells with different phenotypic characteristics is crucial to the discovery of novel cancer drug targets as well as elucidating the basic biologic processes of cancer. However, cell surface proteomics are complex and technologically challenging, and no ideal method is currently available. Here, we describe a strategy that allows scanning of the entire cell surface and identification of molecules that exhibit altered expression between two cell types. Concurrently, this method gives rise to valuable reagents for further characterization of the identified proteins. The strategy is based on subtractive immunization of mice, and we used the two isogenic cell lines, NM-2C5 and M-4A4, derived from the MDA-MB-435 cancer cell line, as a model system. Although the two cell lines are equally tumorigenic, only M-4A4 has metastatic capabilities. Our results yielded a large panel of monoclonal antibodies (mAbs) that recognized cell surface markers preferentially or exclusively expressed on metastatic vs nonmetastatic cancer cells. Four mAbs and their corresponding antigens were further characterized. Importantly, analysis on an extended panel of breast cancer cell lines demonstrated that the four mAbs bound preferentially to cell lines known to be metastatic in vivo, suggesting that these markers have general applications. Immunohistochemical analysis showed that mAb 11E6 reacted preferentially with neuroendocrine tumors while exhibiting no or very weak reactivity with normal tissues. mAb 15C7 stained a variety of cancers as well as some normal lymphoid organs and was subsequently identified to react with HLA-DR-beta. A third mAb, 31D7, that also specifically recognized HLA-DR-beta was capable of inhibiting the growth of MZ2 melanoma cells in vitro. Further, we found that the reduced expression of HLA-DR antigens in nonmetastatic cells of this model was not regulated by class II transactivator, but by posttranscriptional mechanisms. The study demonstrates the advantage of using the exquisitely discriminating recognition system of the immune system itself to scan the cell surface proteome for differentially expressed proteins. The subtractive immunization strategy should be broadly applicable as a quantitative and comparative proteomic approach for screening the cell surface and also allow generation of mAbs to study these cell surface antigens in more detail.

Structural characterization of bimetallic nanomaterials with overlapping x-ray absorption edges

We describe a data analysis method for extended x-ray absorption fine structure spectroscopy suitable for use with compounds of diverse form that contain overlapping absorption edges. This method employs direct concurrent analysis of the data---demonstrated here for cases involving two interfering metal edges---and does not utilize subtractive or data filtering strategies that have been previously used to address this challenge. Its generality and precision are demonstrated in analyses made on two model nanoscale samples: (1) a Ir-Pt nanoparticle system supported on $\ensuremath{\gamma}{\text{-Al}}_{2}{\text{O}}_{3}$ and (2) a hybrid system of Pt nanowires on which Au nanoparticles have been nucleated and grown at the nanowire tips, stacking faults, and twinning boundaries. The results obtained demonstrate the unique compositional and structural qualities of these two systems as well as the broader utility of the new x-ray absorption spectroscopy based protocol used to characterize them.

Of primordial genomes and cooperative kittens

Since every cell is derived from another cell, and the genetic code is more or less universal – so that life cannot, within the bounds of reasonable probability, have evolved more than once – every living being must ultimately derive from an ancestral cell containing a primordial genome. The recognition of the very large part played by horizontal gene transfer in the evolution of bacterial genomes has, however, lately (and notoriously) undermined any hope we may have cherished of tracing the branches of our genomic ancestry back to their prokaryotic roots. In this issue of Journal of Biology Eugene Koonin and colleagues [1] describe an analysis of phylogenetic trees for 6,901 bacterial genes on the basis of which they conclude that, ancestral gene-swapping notwithstanding, a vertical signal (sic) can in fact be discerned at the deepest levels in the phylogenetic tree, though it may never be possible to trace the branches. The extraction of tree structures from the web of gene transfers requires that transferred genes be subtracted by some means from the database of genes used to construct the trees. In the minireview accompanying the paper, Kristen Swithers, Peter Gogarten and Gregory Fournier [2] explain the philosophies and hazards of the strategies for such subtraction, which include the danger of false vertical signals reflecting preferential gene transfer between bacterial species from quite separate branches of the phylogenetic tree and that happen to share a habitat [3]; and the distinct approach whereby Puigbo et al. [1] sought to circumvent the problems of finding the true tree in the thicket. Whether because of horizontal gene transfer or the compression of branching events early in the evolution of prokaryotes, the lines of vertical descent derived by Puigbo et al. from their analysis defy resolution, at least for now and perhaps for ever. There is a character in the comic opera The Mikado, by WS Gilbert and Arthur Sullivan, who claims: 'I can trace my ancestry to a protoplasmal primordial atomic globule. Consequently my family pride is something inconceivable.' Inconceivable and probably misplaced, it would seem. The character is named, more appropriately even than Gilbert could have imagined, Pooh-Bah. The QA but they may find, if they read Ferrell's beautifully navigated expedition through the possible and probable behavior of the subunits of haemoglobin, antibody binding to viral cell surfaces, and the tuning of signal-transducing G proteins, that cooperativity is more complicated and more interesting than they had realized. And the kittens? Sleeping kittens are invoked to explain the Monod-Wyman-Changeux model for cooperative binding of oxygen by hemoglobin, in which it is assumed that oxygen binding to one subunit has no effect on the affinity of the other subunits for oxygen, but that the conformational changes that increase or decrease oxygen affinity occur in unison. Readers who find the behavior of kittens easier to understand than the behavior of molecules may be encouraged by the analogy to read the non-kitten paragraphs too. (I recommend this.) Purists who have no need of kittens will be gratified to note that allostery is implicitly defined by Ferrell as a conformational change at one site in a molecule induced by ligand binding at another site, and not, as in common usage, simply as a change in the conformation of the molecule.

Analysis of expression in the Anopheles gambiae developing testes reveals rapidly evolving lineage-specific genes in mosquitoes

BackgroundMale mosquitoes do not feed on blood and are not involved in delivery of pathogens to humans. Consequently, they are seldom the subjects of research, which results in a very poor understanding of their biology. To gain insights into male developmental processes we sought to identify genes transcribed exclusively in the reproductive tissues of male Anopheles gambiae pupae.ResultsUsing a cDNA subtraction strategy, five male-specifically or highly male-biased expressed genes were isolated, four of which remain unannotated in the An. gambiae genome. Spatial and temporal expression patterns suggest that each of these genes is involved in the mid-late stages of spermatogenesis. Their sequences are rapidly evolving; however, two genes possess clear homologs in a wide range of taxa and one of these probably acts in a sperm motility control mechanism conserved in many organisms, including humans. The other three genes have no match to sequences from non-mosquito taxa, thus can be regarded as orphans. RNA in situ hybridization demonstrated that one of the orphans is transcribed in spermatids, which suggests its involvement in sperm maturation. Two other orphans have unknown functions. Expression analysis of orthologs of all five genes indicated that male-biased transcription was not conserved in the majority of cases in Aedes and Culex.ConclusionDiscovery of testis-expressed orphan genes in mosquitoes opens new prospects for the development of innovative control methods. The orphan encoded proteins may represent unique targets of selective anti-mosquito sterilizing agents that will not affect non-target organisms.

Molecular insights into evolution of the vertebrate gut: focus on stomach and parietal cells in the marsupial, Macropus eugenii

Gastrulation in vertebrate embryos results in the formation of the primary germ layers: ectoderm, mesoderm and endoderm, which contain the progenitors of the tissues of the entire fetal body. Extensive studies undertaken in Xenopus, zebrafish and mouse have revealed a high degree of conservation in the genes and cellular mechanisms regulating endoderm formation. Nodal, Mix and Sox gene factor families have been implicated in the specification of the endoderm across taxa. Considerably less is known about endoderm development in marsupials. In this study we review what is known about the molecular aspects of endoderm development, focusing on evolution and development of the stomach and parietal cells and highlight recent studies on parietal cells in the stomach of Tammar Wallaby, Macropus eugenii. Although the regulation of parietal cells has been extensively studied, very little is known about the regulation of parietal cell differentiation. Intriguingly, during late-stage forestomach maturation in M. eugenii, there is a sudden and rapid loss of parietal cells, compared with the sharp increase in parietal cell numbers in the hindstomach region. This has provided a unique opportunity to study the development and regulation of parietal cell differentiation. A PCR-based subtractive hybridization strategy was used to identify candidate genes involved in this phenomenon. This will allow us to dissect the molecular mechanisms that underpin regulation of parietal cell development and differentiation, which have been a difficult process to study and provide markers that can be used to study the evolutionary origin of these cells in vertebrates.

Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells

Embryonic stem cells (ESCs) and embryonic germ cells (EGCs) provide exciting models for understanding the underlying mechanisms that make a cell pluripotent. Indeed, such understanding would enable dedifferentiation and reprogrammation of any cell type from a patient needing a cell therapy treatment. Proteome analysis has emerged as an important technology for deciphering these biological processes and thereby ESC and EGC proteomes are increasingly studied. Nevertheless, their nuclear proteomes have only been poorly investigated up to now. In order to investigate signaling pathways potentially involved in pluripotency, proteomic analyses have been performed on mouse ESC and EGC nuclear proteins. Nuclei from ESCs and EGCs at undifferentiated stage were purified by subcellular fractionation. After 2-D separation, a subtractive strategy (subtracting culture environment contaminating spots) was applied and a comparison of ESC, (8.5 day post coïtum (dpc))-EGC and (11.5 dpc)-EGC specific nuclear proteomes was performed. A total of 33 ESC, 53 (8.5 dpc)-EGC, and 36 (11.5 dpc)-EGC spots were identified by MALDI-TOF-MS and/or nano-LC-MS/MS. This approach led to the identification of two isoforms (with and without N-terminal acetylation) of a known pluripotency marker, namely developmental pluripotency associated 5 (DPPA5), which has never been identified before in 2-D gel-MS studies of ESCs and EGCs. Furthermore, we demonstrated the efficiency of our subtracting strategy, in association with a nuclear subfractionation by the identification of a new protein (protein arginine N-methyltransferase 7; PRMT7) behaving as proteins involved in pluripotency.

Lung-selective gene responses to alveolar hypoxia: potential role for the bone morphogenetic antagonist gremlin in pulmonary hypertension

Pulmonary hypoxia is a common complication of chronic lung diseases leading to the development of pulmonary hypertension. The underlying sustained increase in vascular resistance in hypoxia is a response unique to the lung. Thus we hypothesized that there are genes for which expression is altered selectively in the lung in response to alveolar hypoxia. Using a novel subtractive array strategy, we compared gene responses to hypoxia in primary human pulmonary microvascular endothelial cells (HMVEC-L) with those in cardiac microvascular endothelium and identified 90 genes (forming 9 clusters) differentially regulated in the lung endothelium. From one cluster, we confirmed that the bone morphogenetic protein (BMP) antagonist, gremlin 1, was upregulated in the hypoxic murine lung in vivo but was unchanged in five systemic organs. We also demonstrated that gremlin protein was significantly increased by hypoxia in vivo and inhibited HMVEC-L responses to BMP stimulation in vitro. Furthermore, significant upregulation of gremlin was measured in lungs of patients with pulmonary hypertensive disease. From a second cluster, we showed that CXC receptor 7, a receptor for the proangiogenic chemokine CXCL12, was selectively upregulated in the hypoxic lung in vivo, confirming that our subtractive strategy had successfully identified a second lung-selective hypoxia-responsive gene. We conclude that hypoxia, typical of that encountered in pulmonary disease, causes lung-specific alterations in gene expression. This gives new insights into the mechanisms of pulmonary hypertension and vascular loss in chronic lung disease and identifies gremlin 1 as a potentially important mediator of vascular changes in hypoxic pulmonary hypertension.