Jason L. Herring

Inhaled Nitrite Reverses Hemolysis-Induced Pulmonary Vasoconstriction in Newborn Lambs Without Blood Participation

Background— Nitrite can be converted to nitric oxide (NO) by a number of different biochemical pathways. In newborn lambs, an aerosol of inhaled nitrite has been found to reduce pulmonary blood pressure, possibly acting via conversion to NO by reaction with intraerythrocytic deoxyhemoglobin. If so, the vasodilating effects of nitrite would be attenuated by free hemoglobin in plasma that would rapidly scavenge NO. Methods and Results— Pulmonary vascular pressures and resistances to flow were measured in anesthetized newborn lambs. Plasma hemoglobin concentrations were then elevated, resulting in marked pulmonary hypertension. This effect was attenuated if infused hemoglobin was first oxidized to methemoglobin, which does not scavenge NO. These results further implicate NO as a tonic pulmonary vasodilator. Next, while free hemoglobin continued to be infused, the lambs were given inhaled NO gas (20 ppm), inhaled sodium nitrite aerosol (0.87 mol/L), or an intravascular nitrite infusion (3 mg/h bolus, 5 mg · kg−1 · h−1 infusion). Inhaled NO and inhaled nitrite aerosol both resulted in pulmonary vasodilation. Intravascular infusion of nitrite, however, did not. Increases in exhaled NO gas were observed in lambs while breathing the nitrite aerosol (≈20 ppb NO) but not during intravascular infusion of nitrite. Conclusions— We conclude that the pulmonary vasodilating effect of inhaled nitrite results from its conversion to NO in airway and parenchymal lung tissue and is not dependent on reactions with deoxyhemoglobin in the pulmonary circulation. Inhaled nitrite aerosol remains a promising candidate to reduce pulmonary hypertension in clinical application.

Mass Spectrometric Studies on Epigenetic Interaction Networks in Cell Differentiation

Arrest of cell differentiation is one of the leading causes of leukemia and other cancers. Induction of cell differentiation using pharmaceutical agents has been clinically attempted for the treatment of these cancers. Epigenetic regulation may be one of the underlying molecular mechanisms controlling cell proliferation or differentiation. Here, we report on the use of proteomics-based differential protein expression analysis in conjunction with quantification of histone modifications to decipher the interconnections among epigenetic modifications, their modifying enzymes or mediators, and changes in the associated pathways/networks that occur during cell differentiation. During phorbol-12-myristate 13-acetate-induced differentiation of U937 cells, fatty acid synthesis and its metabolic processing, the clathrin-coated pit endocytosis pathway, and the ubiquitin/26 S proteasome degradation pathways were up-regulated. In addition, global histone H3/H4 acetylation and H2B ubiquitination were down-regulated concomitantly with impaired chromatin remodeling machinery, RNA polymerase II complexes, and DNA replication. Differential protein expression analysis established the networks linking histone hypoacetylation to the down-regulated expression/activity of p300 and linking histone H2B ubiquitination to the RNA polymerase II-associated FACT-RTF1-PAF1 complex. Collectively, our approach has provided an unprecedentedly systemic set of insights into the role of epigenetic regulation in leukemia cell differentiation.

Inhaled Nitric Oxide Therapy Increases Blood Nitrite, Nitrate, and S-Nitrosohemoglobin Concentrations in Infants with Pulmonary Hypertension

OBJECTIVE To measure the circulating concentrations of nitric oxide (NO) adducts with NO bioactivity after inhaled NO (iNO) therapy in infants with pulmonary hypertension. STUDY DESIGN In this single center study, 5 sequential blood samples were collected from infants with pulmonary hypertension before, during, and after therapy with iNO (n = 17). Samples were collected from a control group of hospitalized infants without pulmonary hypertension (n = 16) and from healthy adults for comparison (n = 12). RESULTS After beginning iNO (20 ppm) whole blood nitrite levels increased approximately two-fold within 2 hours (P<.01). Whole blood nitrate levels increased to 4-fold higher than baseline during treatment with 20 ppm iNO (P<.01). S-nitrosohemoglobin increased measurably after beginning iNO (P<.01), whereas iron nitrosyl hemoglobin and total hemoglobin-bound NO-species compounds did not change. CONCLUSION Treatment of pulmonary hypertensive infants with iNO results in increases in levels of nitrite, nitrate, and S-nitrosohemoglobin in circulating blood. We speculate that these compounds may be carriers of NO bioactivity throughout the body and account for peripheral effects of iNO in the brain, heart, and other organs.

Epigenetics of chronic rhinosinusitis and the role of the eosinophil

One theory for the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) involves aberration in the expression of genes that maintain the sinonasal innate immune system. We propose that the alteration in gene expression seen in CRSwNP is a result of oxidative byproducts of eosinophils. Activated eosinophils and neutrophils may lead to the production of hypobromous acid (HOBr) and hypochlorous acid (HOCL) and the posttranslational modification products 5‐bromocytosine (5BrC) and 5‐chlorocytosine (5ClC), respectively. 5BrC and 5ClC may cause aberrant methylation of cytosine during DNA replication and mimic the endogenous methylation signal associated with gene silencing. We propose to use gas chromatography–mass spectrometry (GC‐MS) to identify the presence of 5BrC and 5ClC in CRSwNP patients.

Enzymatic methylation of DNA in cultured human cells studied by stable isotope incorporation and mass spectrometry.

Enzymatic methylation of cytosine residues in DNA, in conjunction with covalent histone modifications, establishes an epigenetic code essential for the proper control of gene expression in higher organisms. Once established during cellular differentiation, the epigenetic code must be faithfully transmitted to progeny cells. However, epigenetic perturbations can be found in most if not all cancer cells, and the mechanisms leading to these changes are not well understood. In this paper, we describe a series of experiments aimed at understanding the dynamic process of DNA methylation that follows DNA replication. Cells in culture can be propagated in the presence of (15)N-enriched uridine, which labels the pyrimidine precursor pool as well as newly replicated DNA. Simultaneous culture in the presence of (2)H-enriched methionine results in labeling of newly methylated cytosine residues. An ensemble of 5-methylcytosine residues differing in the degree of isotopic enrichment is generated, which can be examined by mass spectrometry. Using this method, we demonstrate that the kinetics of both DNA replication and methylation of newly replicated DNA are indistinguishable. The majority of methylation following DNA replication is shown to occur on the newly synthesized DNA. The method reported here does, however, suggest an unexpected methylation of parental DNA during DNA replication, which might indicate a previously undescribed chromatin remodeling process. The method presented here will be useful in monitoring the dynamic process of DNA methylation and will allow a more detailed understanding of the mechanisms of clinically used methylation inhibitors and environmental toxicants.

Measurement of Postreplicative DNA Metabolism and Damage in the Rodent Brain

The DNA of all organisms is metabolically active due to persistent endogenous DNA damage, repair, and enzyme-mediated base modification pathways important for epigenetic reprogramming and antibody diversity. The free bases released from DNA either spontaneously or by base excision repair pathways constitute DNA metabolites in living tissues. In this study, we have synthesized and characterized the stable-isotope standards for a series of pyrimidines derived from the normal DNA bases by oxidation and deamination. We have used these standards to measure free bases in small molecule extracts from rat brain. Free bases are observed in extracts, consistent with both endogenous DNA damage and 5-methylcytosine demethylation pathways. The most abundant free base observed is uracil, and the potential sources of uracil are discussed. The free bases measured in tissue extracts constitute the end product of DNA metabolism and could be used to reveal metabolic disturbances in human disease.

Probes and Targets of DNA Methylation and Demethylation in Drug Development

DNA methylation and demethylation is part of the essential biological processes regulating gene expression in normal cell development. Abnormal methylation status of specific genes and their irregularly translated proteins are normally associated with certain kinds of diseases or cancer. The rapid development of innovative DNA methylation mapping techniques provides a better understanding of DNA methylation pattern and its mechanisms in the human genome and its correlation with numerous diseases. These new techniques can lead us to develop new epigenetic medications, such as DNA methyltransferase inhibitors. As part of the approaches to probe DNA methylation and evaluate the effects of epigenetic therapy, mass spectrometry has been taking an important role in the identification, validation, and quantification of DNA methylation and demethylation. In this review, we will briefly summarize the current breadth of knowledge on the topic of DNA methylation and its occurrence in diseases, DNA methylation drugs, and mass spectrometry based approaches used to study DNA methylation.

Analysis of glucose-derived amino acids involved in one-carbon and cancer metabolism by stable-isotope labeling and gas chromatography mass spectrometry

A major hallmark of cancer is a perturbed metabolism resulting in high demand for various metabolites, glucose being the most well studied. While glucose can be converted into pyruvate for ATP production, the serine synthesis pathway (SSP) can divert glucose to generate serine, glycine, and methionine. In the process, the carbon unit from serine is incorporated into the one-carbon pool which makes methionine and maintains S-adenosylmethionine levels, which are needed to maintain the epigenetic landscape and ultimately controlling what genes are available for transcription. Alternatively, the carbon unit can be used for purine and thymidylate synthesis. We present here an approach to follow the flux through this pathway in cultured human cells using stable isotope enriched glucose and gas chromatography mass spectrometry analysis of serine, glycine, and methionine. We demonstrate that in three different cell lines this pathway contributes only 1-2% of total intracellular methionine. This suggests under high extracellular methionine conditions, the predominance of carbon units from this pathway are used to synthesize nucleic acids.

Proteome Analysis of Hypoxic Glioblastoma Cells Reveals Sequential Metabolic Adaptation of One-Carbon Metabolic Pathways

Rapidly proliferating tumors are exposed to a hypoxic microenvironment because of their density, high metabolic consumption, and interruptions in blood flow because of immature angiogenesis. Cellular responses to hypoxia promote highly malignant and metastatic behavior, as well as a chemotherapy-resistant state. To better understand the complex relationships between hypoxic adaptations and cancer progression, we studied the dynamic proteome responses of glioblastoma cells exposed to hypoxia via an innovative approach: quantification of newly synthesized proteins using heavy stable-isotope arginine labeling combined with accurate assessment of cell replication by quantification of the light/heavy arginine ratio of peptides in histone H4. We found that hypoxia affects cancer cells in multiple intertwined ways: inflammation, typically with over-expressed glucose transporter (GLUT1), DUSP4/MKP2, and RelA proteins; a metabolic adaptation with overexpression of all glycolytic pathway enzymes for pyruvate/lactate synthesis; and the EMT (epithelial-mesenchymal transition) and cancer stem cell (CSC) renewal with characteristic morphological changes and mesenchymal/CSC protein expression profiles. For the first time, we identified the vitamin B12 transporter protein TCN2, which is essential for one-carbon metabolism, as being significantly downregulated. Further, we found, by knockdown and overexpression experiments, that TCN2 plays an important role in controlling cancer cell transformation toward the highly aggressive mesenchymal/CSC stage; low expression of TCN2 has an effect similar to hypoxia, whereas high expression of TCN2 can reverse it. We conclude that hypoxia induces sequential metabolic responses of one-carbon metabolism in tumor cells. Our mass spectrometry data are available via ProteomeXchange with identifiers PXD005487 (TMT-labeling) and PXD007280 (label-free).

Inhaled nitrite reverses hemolysis-induced pulmonary vasoconstriction in newborn lambs without blood participation: Blood- Conversion of inhaled nitrite to NO

Background— Nitrite can be converted to nitric oxide (NO) by a number of different biochemical pathways. In newborn lambs, an aerosol of inhaled nitrite has been found to reduce pulmonary blood pressure, possibly acting via conversion to NO by reaction with intraerythrocytic deoxyhemoglobin. If so, the vasodilating effects of nitrite would be attenuated by free hemoglobin in plasma that would rapidly scavenge NO. Methods and Results— Pulmonary vascular pressures and resistances to flow were measured in anesthetized newborn lambs. Plasma hemoglobin concentrations were then elevated, resulting in marked pulmonary hypertension. This effect was attenuated if infused hemoglobin was first oxidized to methemoglobin, which does not scavenge NO. These results further implicate NO as a tonic pulmonary vasodilator. Next, while free hemoglobin continued to be infused, the lambs were given inhaled NO gas (20 ppm), inhaled sodium nitrite aerosol (0.87 mol/L), or an intravascular nitrite infusion (3 mg/h bolus, 5 mg · kg−1 · h−1 infusion). Inhaled NO and inhaled nitrite aerosol both resulted in pulmonary vasodilation. Intravascular infusion of nitrite, however, did not. Increases in exhaled NO gas were observed in lambs while breathing the nitrite aerosol (≈20 ppb NO) but not during intravascular infusion of nitrite. Conclusions— We conclude that the pulmonary vasodilating effect of inhaled nitrite results from its conversion to NO in airway and parenchymal lung tissue and is not dependent on reactions with deoxyhemoglobin in the pulmonary circulation. Inhaled nitrite aerosol remains a promising candidate to reduce pulmonary hypertension in clinical application.