Effects of different immunogenic amino acids in PD-L1 vaccine on the differentiation of T cell subsets / 中国药科大学学报

@#To compare the effects of endogenous 3-nitrotyrosine and non-natural 4-nitrophenylalanine in PD-L1 vaccine on the differentiation of T cell subsets, two immunogenic amino acids were introduced into the same site of PD-L1 vaccine. Two PD-L1 mutants with 3-nitrotyrosine and 4-nitrophenylalanine were obtained, respectively, using genetic code expansion technology. Mice were immunized with these two mutants, and their effects on the differentiation of T cell subsets in spleen were analyzed. The results of flow cytometry showed that the introduction of 4-nitrophenylalanine in PD-L1 vaccine could promote the polarization of Th1 cells while reducing the proportion of Treg cells; the introduction of 3-nitrotyrosine had no effect on the polarization of Th1 cells, while significantly increasing the proportion of Treg and Th17 cells. The introduction of both into PD-L1 vaccine could promote the response of CD8+ T cells in spleen, and the response of PD-L1 mutant containing 4-nitrophenylalanine was stronger. In summary, the non-natural 4-nitrophenylalanine is more suitable for the design of tumor vaccines as compared with endogenous 3-nitrotyrosine.

Association of serum 3-nitrotyrosine with carotid atherosclerosis in newly diagnosed type 2 diabetic patients / 中华内分泌代谢杂志

To investigate the association of serum 3-nitrotyrosine ( 3-NT ) with carotid atherosclerosis in newly diagnosed type 2 diabetic patients. 96 patients with newly diagnosed type 2 diabetes mellitus treated in the department of endocrinology of Tangshan Gongren Hospital were recruited, and were divided into two groups depending on their carotid atherosclerosis status as carotid atherosclerosis group ( CAS group, n = 54 ) and non-carotid atherosclerosis group ( NCAS group, n=42); while 51 healthy subjects without type 2 diabetes mellitus from the medical examination center were recruited as normal control group ( NC group, n=51) . Demographic and clinical data of all subjects were collected. Serum 3-NT levels were measured by Enzyme-Linked Immunosorbent Assay ( ELISA) . ( 1) The levels of 3-NT in CAS group and NCAS group were all higher than those in NC group, and the level of 3-NT in CAS group was higher than that in NCAS group (all P<0.05);(2) In type 2 diabetic patients, 3-NT was positively correlated with HbA1C and low density lipoprotein-cholesterol ( both P<0. 05); ( 3) Logistic regression analysis showed that age (OR=1.271, P=0.023), HbA1C(OR=1.812, P=0.005) , Hcy (OR=1.194, P=0.019), and 3-NT (OR=1.593, P=0.011) were risk factors of CAS in type 2 diabetic patients. Serum 3-NT was closely correlated with carotid atherosclerosis in newly diagnosed type 2 diabetic patients, suggesting that serum 3-NT may be involved in the carotid atherosclerosis of newly diagnosed type 2 diabetic patients.

The inhibitory effect of PNS on brain 3-NT formation in vitro / 天津医药

Objective To study the inhibitory effect of panax notoginseng saponins (PNS) on 3-nitrotyrosine (3-NT) formation in brain induced by heme/NO2 -/H2O2 or ONOO - pathways in vitro. Methods According to the two major pathways of 3-NT formation in vivo, the models of protein nitration induced by heme/NaNO2/H2O2 or ONOO-system were established, respectively, in vitro. Bovine serum albumin (BSA)/rat plasma protein or rat brain homogenate protein were utilized as reactive substrates in both systems. Samples were divided into blank-control group, 3-NT group and PNS group (including low-, medium-and high-concentration subgroups). In 3-NT group, samples were exposed to heme/NaNO2/H2O2 or ONOO-system, respectively, at 37℃for 30 min, whereas in PNS group, samples were pre-incubated with PNS (at final concentrations of 50 mg/L, 100 mg/L, and 200 mg/L) at 37℃for 5 min before the nitrating system exposure. The 3-NT level in each group was detected by Western blot assy. Results Compared with the blank-control group, both heme/NaNO2/H2O2 and ONOO-system can induce significant 3-NT generation in BSA/rat plasma protein or rat brain homogenate protein (P0.05). Medium- and high-concentrations of PNS pre-treatment markedly inhibited 3-NT accumulation, with maximum effect at the concentration of 200 mg/L (P<0.05). Conclusion Medium- and high-concentrations of PNS can inhibit 3-NT formation in brain tissue mediated by either heme/NO2-/H2O2 or ONOO-pathways, implying that potential neuroprotective action against 3-NT involves pathological conditions, like trauma, stroke, and neurodegenerative diseases.

Sodium nitroprusside has leishmanicidal activity independent of iNOS

Abstract: INTRODUCTION: Leishmaniasis is a zoonotic disease caused by protozoa of the genus Leishmania . Cutaneous leishmaniasis is the most common form, with millions of new cases worldwide each year. Treatments are ineffective due to the toxicity of existing drugs and the resistance acquired by certain strains of the parasite. METHODS: We evaluated the activity of sodium nitroprusside in macrophages infected with Leishmania (Leishmania) amazonensis . Phagocytic and microbicidal activity were evaluated by phagocytosis assay and promastigote recovery, respectively, while cytokine production and nitrite levels were determined by ELISA and by the Griess method. Levels of iNOS and 3-nitrotyrosine were measured by immunocytochemistry. RESULTS: Sodium nitroprusside exhibited in vitro antileishmanial activity at both concentrations tested, reducing the number of amastigotes and recovered promastigotes in macrophages infected with L. amazonensis . At 1.5µg/mL, sodium nitroprusside stimulated levels of TNF-α and nitric oxide, but not IFN-γ. The compound also increased levels of 3-nitrotyrosine, but not expression of iNOS, suggesting that the drug acts as an exogenous source of nitric oxide. CONCLUSIONS: Sodium nitroprusside enhances microbicidal activity in Leishmania -infected macrophages by boosting nitric oxide and 3-nitrotyrosine.

Effects of enos uncoupling on the endothelial dysfunction of pulmonary artery in rats with decompression sickness / 解放军医学杂志

Objective To explore the effects of unsafe decompression on the endothelial function of pulmonary artery in rat and its possible related mechanism. Methods Sixty male SD rats (260±35g) were randomly divided into two groups (30 each): control group and decompression (DCS) group. Decompression sickness (DCS) model was reproduced by placing the rats in a compression chamber with air pressure of 600kPa for 60min, followed by decompression at a rate of 100kPa/min to normal pressure. The surviving rats in both control and DCS groups were sacrificed and their pulmonary artery was harvested. The endothelium dependent vasodilatation capacity of isolated pulmonary artery was assessed. The expression and uncoupling of endothelial nitric oxide synthetase (eNOS), as well as the nitration level of each kind of protein in the pulmonary artery tissue, were analyzed by Western blotting. The concentration of reactive oxygen species (ROS) in the pulmonary artery was determined with superoxide anion probe dihematoporphyrin ether (DHE) staining. Results Ten of 30 rats in DC group died of unsafe decompression, and the endothelium dependent vasodilatation capacity of excised pulmonary artery in survived rats was found to decline obviously (P0.05), but the ratio of eNOS monomer/dimer increased significantly in DC group than in control group (P<0.05). The tyrosine nitration level of each kind of protein in the pulmonary artery tissues was higher significantly in DC group than that in control group (P<0.05). DHE showed that the generated amount of DCS in pulmonary artery tissues was obviously higher in DC group than in control group (P<0.05). Conclusions Unsafe decompression may lead to uncoupling of eNOS dimers in the endothelium of pulmonary artery. Uncoupled eNOS monomers may inhibit the synthesis of NO, thereby affect the endothelium dependent vasodilatation function. On the other hand, the eNOS monomers may facilitate the anabolism of ONOO-, leading to an increase in tyrosine nitration level of each kind of protein in the pulmonary artery tissues, thereby cause the regulation disorder of cell information system. The eNOS monomers may also increase the production of ROC, there by mediate the peroxide injuries.

Simultaneous determination of 3-chlorotyrosine and 3-nitrotyrosine in human plasma by direct analysis in real time-tandem mass spectrometry

A novel method for the simultaneous determination of 3-nitrotyrosine (NT) and 3-chlorotyrosine (CT) in human plasma has been developed based on direct analysis in real time-tandem mass spectrometry (DART-MS/MS). Analysis was performed in the positive ionization mode using multiple reaction monitoring (MRM) of the ion transitions at m/z 216.2/170.1 for CT, m/z 227.2/181.1 for NT and m/z 230.2/184.2 for the internal standard, d (3)-NT. The assay was linear in the ranges 0.5-100 μg/mL for CT and 4-100 μg/mL for NT with corresponding limits of detection of 0.2 and 2 μg/mL. Intra- and inter-day precisions and accuracies were respectively <15% and ±15%. Matrix effects were also evaluated. The method is potentially useful for high throughput analysis although sensitivity needs to be improved before it can be applied in clinical research.

Malondialdehyde and 3-Nitrotyrosine in Exhaled Breath Condensate in Retired Elderly Coal Miners with Chronic Obstructive Pulmonary Disease

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an important cause of occupational mortality in miners exposed to coal mine dust. Although the inflammatory mediators involved in COPD have not been defined, many studies have shown that inflammatory mediators such as reactive oxygen and nitrogen species are involved in orchestrating the complex inflammatory process in COPD. METHODS: To investigate the relevance of exhaled biomarkers of oxidative and nitrosative stress in participants with COPD, we determined the levels of hydrogen peroxide, malondialdehyde (MDA), and 3-nitrotyrosine (3-NT) in exhaled breath condensate (EBC) in 90 retired elderly coal miners (53 non-COPD and 37 COPD participants). RESULTS: Mean levels of MDA (4.64 nM vs. 6.46 nM, p = 0.005) and 3-NT (3.51 nM vs. 5.50 nM, p = 0.039) in EBC were significantly higher in participants with COPD. The median level of MDA did show statistical difference among the COPD severities (p = 0.017), and the area under the receiver operating characteristic curve for MDA (0.67) for the diagnostic discrimination of COPD indicated the biomarker. The optimal cutoff values were 5.34 nM (64.9% sensitivity and 64.2% specificity) and 5.58 nM (62.2% sensitivity and 62.3% specificity) for MDA and 3-NT, respectively. The results suggest that high levels of MDA and 3-NT in EBC are associated with COPD in retired elderly miners. CONCLUSION: These results showed that the elevated levels of EBC MDA and EBC 3-NT in individuals with COPD are biomarkers of oxidative or nitrosative stress.

Tyrosine nitration site specificity identified by LC/MS in nitrite-modified collagen type IV

Non-enzymatic nitrite induced collagen cross-linking results in changes reminiscent of age-related damage and parallels the well-known model system, non-enzymatic glycation. We have recently observed that nitrite modification of basement membrane proteins can induce deleterious effects on overlying retinal pigment epithelial cells in studies relevant to age-related macular degeneration. The present work was undertaken in order to confirm 3-nitro-tyrosine (3-NT) as a product of the reaction and to identify the site specificity of nitration in collagen IV, a major component of basement membranes. Human collagen type IV was modified via incubation with 200 mM NaNO2 (pH=7.38) for one week at 37degrees C. The modified protein was prepared in 2 different ways, including acid hydrolysis and trypsin digestion for site specificity determination. The samples were analyzed by LC/MS using a C12 RP column. Site specificity was determined from tandem MS/MS data utilizing TurboSEQUEST software and the Swiss-Prot sequence database. 3-NT was detected in protein digests and acid hydrolysates of nitrite modified collagen IV. Positive identification with standard 3-NT was confirmed by identical Rt, lambda(max)=279 nm and 355 nm, and m/z=227. Analyses of tryptic digests identified four sites of tyrosine nitration, alpha1(IV)Y348, alpha1(IV)Y534, alpha2(IV)Y327, and alpha2(IV)Y1081. These sites are located in the triple-helical region of the protein and provide clues regarding potential sites for nitrite modification in collagen type IV.