Gold nanoclusters-based fluorescence resonance energy transfer for rapid and sensitive detection of Pb2.

Lead pollution has remained a significant global concern for several decades due to its detrimental effects on the brain, heart, kidneys, lungs, and immune system across all age groups. Addressing the demand for detecting trace amounts of lead in food samples, we have developed a novel biosensor based on fluorescence resonance energy transfer (FRET) from fluorescein R6G to gold nanoclusters (AuNCs-CCY). By utilizing polypeptides as a template, we successfully synthesized AuNCs-CCY with an excitation spectrum that overlaps with the emission spectrum of R6G. Exploiting the fact that Pb2+ induces the aggregation of gold nanoclusters, leading to the separation of R6G from AuNCs-CCY and subsequent fluorescence recovery, we achieved the quantitative detection of Pb2+. Within the concentration range of 0.002-0.20 µM, a linear relationship was observed between the fluorescence enhancement value (F-F0) and Pb2+ concentration, characterized by the linear equation y = 2398.69x + 87.87 (R2 = 0.996). The limit of detection (LOD) for Pb2+ was determined to be 0.00079 µM (3σ/K). The recovery rate ranged from 96 % to 104 %, with a relative standard deviation (RSD) below 10 %. These findings demonstrate the potential application value of our biosensor, which offers a promising approach to address the urgent need for sensitive detection of heavy metal ions, specifically Pb2+, in food samples.

A ratio fluorescence method based on dual emissive gold nanoclusters for detection of biomolecules and metal ions.

Gold nanoclusters have good biocompatibility and can be easily modified to improve their luminescence properties. In this study, we prepared a new type of dual-emitting gold nanoclusters (d-Au NCs) for discriminative detection of phenylalanine and Fe3+ with high selectivity and sensitivity. The fluorescence sensor which was synthesized without any further assembly or conjugation shows dual-emissions at 430 nm and 600 nm under a single excitation at 350 nm. Phenylalanine can turn on the red emission of the probe, while Fe3+ can turn on its yellow emission and turn off the red emission. By detecting a variety of amino acids and metal ions, d-Au NCs showed good selectivity to phenylalanine and Fe3+. Finally, this method was applied to determine phenylalanine and Fe3+ in lake water, human urine and milk, which has certain application prospects in the field of biology and environment.

Extraction and detection of morin from Sanghuangporus lonicericola by magnetic molecularly imprinted polymers coupled with HPLC analysis.

In this study, morin magnetic molecularly imprinted polymers (Morin-MMIPs) were synthesized based on magnetic nanoparticles and surface molecularly imprinted technology with superparamagnetism and extraction selectivity. The polymers allowed the separating of morin from complex matrices in the presence of an external magnetic field with no need for centrifugation or filtration. The microstructure of the polymers was characterized by scanning electron microscopy and transmission electron microscopy. Meanwhile, the functional group and magnetic properties of the polymers were characterized using Fourier transform infrared spectroscopy (FT-IR) and magnetic vibration meter (VSM). The maximum adsorption capacity of MMIPs was 3.24 mg/g, which was 2.55 times higher than that of MNIPs (1.27 mg/g). Morin was quantified by HPLC-DAD, which showed good linearity in the concentration range of 0.05-60 µg/ml with the correlation coefficient R2  = 0.9993. The limit of detection (LOD) was 0.08 µg/ml, and the spiked recoveries were 87.5-106.8%. The calculation of the adsorption isotherm and kinetic model revealed the adsorption mechanisms, and the adsorption process was consistent with the Langmuir adsorption isotherm and pseudo-secondary kinetic models. Likewise, the material has been successfully used to extract and separate morin from food samples. The method reported in this paper has the advantages of fast adsorption speed, high selectivity, and environmental friendliness. It provided a reliable method for the separation and detection of morin or other natural products.

Single-atom sites on perovskite chips for record-high sensitivity and quantification in SERS.

Surface enhanced Raman scattering (SERS) is a rapid and nondestructive technique that is capable of detecting and identifying chemical or biological compounds. Sensitive SERS quantification is vital for practical applications, particularly for portable detection of biomolecules such as amino acids and nucleotides. However, few approaches can achieve sensitive and quantitative Raman detection of these most fundamental components in biology. Herein, a noble-metal-free single-atom site on a chip strategy was applied to modify single tungsten atom oxide on a lead halide perovskite, which provides sensitive SERS quantification for various analytes, including rhodamine, tyrosine and cytosine. The single-atom site on a chip can enable quantitative linear SERS responses of rhodamine (10-6-1 mmol L-1), tyrosine (0.06-1 mmol L-1) and cytosine (0.2-45 mmol L-1), respectively, which all achieve record-high enhancement factors among plasmonic-free semiconductors. The experimental test and theoretical simulation both reveal that the enhanced mechanism can be ascribed to the controllable single-atom site, which can not only trap photoinduced electrons from the perovskite substrate but also enhance the highly efficient and quantitative charge transfer to analytes. Furthermore, the label-free strategy of single-atom sites on a chip can be applied in a portable Raman platform to obtain a sensitivity similar to that on a benchtop instrument, which can be readily extended to various biomolecules for low-cost, widely demanded and more precise point-of-care testing or in-vitro detection. Electronic Supplementary Material: Supplementary material is available for this article at 10.1007/s40843-022-1968-5 and is accessible for authorized users.

Synthesis and characterization of genistein magnetic molecularly imprinted polymers and their application in soy sauce products.

In this study, a novel method based on genistein magnetic molecularly imprinted polymers (Gen-MMIPs) was developed utilizing a surface molecular imprinting technique, in which genistein was used as the template molecule and Fe3O4 was used as the carrier. The synthesis of Gen-MMIPs was characterized by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which indicated that the diameter of the Gen-MMIPs was approximately 500 nm. Via analysis with a vibrating sample magnetometer (VSM), the saturation magnetization of Gen-MMIPs was determined to be 24.79 emu g-1. Fourier transform infrared (FT-IR) spectroscopy showed that polymer groups were on the surface of the magnetic carrier. Adsorption experiments suggested that the genistein adsorption capability of Gen-MMIPs was 5.81 mg g-1, and adsorption equilibrium was achieved within 20 min. Gen-MMIPs as dispersive solid-phase extraction (dSPE) adsorbents combined with HPLC were used to selectively separate genistein in soy sauce samples, and the recoveries ranged from 85.7 to 88.5% with relative standard deviations (RSDs) less than 5%, which proved that this method can be used for the detection of genistein residues in real samples.

[Research progress of AFP in the diagnosis and therapy of hepatocellular carcinoma].

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths and the fifth most common cancer worldwide. Clinically therapeutic options for HCC are very limited, and the overall survival rate of patients is very low. Therefore, early diagnosis and treatment of HCC have important impact on overall survival of patients. At present, alpha-fetoprotein (AFP) is one of the most widely used serological markers for HCC. Many evidences have shown that as a specific onco-protein, AFP has great research value in the occurrence, development, diagnosis and treatment of HCC. Here, we briefly introduce the molecular mechanism of AFP in the regulation of HCC occurrence and development, and its role in tumor escape from immune surveillance. We focus on the application of AFP as an important HCC target or carcino-embryonic antigen (CEA) in HCC clinical diagnosis and treatment.

Risk screening of mycotoxins and their derivatives in dairy products using a stable isotope dilution assay and LC-MS/MS.

An liquid chromatography-tandem mass spectrometry method coupled with a stable isotope dilution assay was established for the simultaneous detection of 17 mycotoxins and their derivatives (aflatoxins B1 , B2 , G1 , G2 , M1 , and M2 ; fumonisins B1 and B2 ; ochratoxin A; zearalenone; zearalanone; α-zearalanol; α-zearalenol; T-2 toxin; deoxynivalenol; deepoxy-deoxynivalenol; and sterigmatocystin) in milk and dairy products. The mycotoxins were extracted with acidified acetonitrile and the lipids were removed using a Captiva EMR-lipid column. The average recoveries of the target compounds from samples spiked at three different concentrations were 67-102%, and the relative standard deviations of the peak areas were less than 10%. Limits of quantification (S/N = 10) of 0.004-1.25 µg/kg were achieved, which are significantly lower than the maximum levels allowed in various countries and regions for each regulated mycotoxin. Milk and yogurt products from local markets and e-commercial platforms were analyzed using the optimized method. The screening showed that aflatoxin M1 , deoxynivalenol, fumonisins B1 and B2 , and zearalenone could be found in milk and yogurt products, especially those products also containing grains or jujube ingredients, indicating that there is a risk of mycotoxins in dairy products.

Pine (Pinus sylvestris L.) bark proanthocyanidins affords prevention of peroxynitrite-induced l-tyrosine nitration, DNA damage and hydroxyl radical formation.

Peroxynitrite is known as a strong deleterious species that may readily trigger several geriatric diseases via injuring cellular constituents. Proanthocyanidins, a biological flavonoids constituent of Pinus sylvestris L. bark, has been attributed a large variety of pharmacological functions to its antioxidant potential. The results revealed that peroxynitrite could cause the generation of hydroxyl radical, the breakage of φX-174 plasmid DNA brand as well as the nitration of L-tyrosine. However, pine (Pinus sylvestris L.) bark proanthocyanidins extracts at low concentration range markedly inhibited the peroxynitrite -induced the formation of open circular DNA form (IC50 = 5.03±0.39 mg/mL). The 3-Nitro-L-tyrosine generated by the reaction of peroxynitrite with L-tyrosine was reduced by PBP (IC50 = 1.01±0.01 mg/mL). Besides, electron spin resonance spectroscopy data indicates that the intensive signal of dimethyl pyridine N-oxide hydroxyl radical adduct from peroxynitrite was reversed by pine bark proanthocyanidins extracts (IC50 =1.02±0.04 mg/mL). Moreover, the obtained data shows that PBP provides more efficient protection against peroxynitrite than that of ascorbic acid. Together, the present study suggests that pine bark proanthocyanidins could exert potent preventive activity against peroxynitrite -elicited cytotoxicity on the biomacromolecules, a study-worthy finding with pharmacological importance.

A peptide-based inhibitor of gp96 suppresses HBsAg expression and HBV replication by upregulation of p53.

In hepatitis B virus (HBV) infection, the virus produces redundant hepatitis B surface antigen (HBsAg) that plays a key role in driving T-cell tolerance and viral persistence. However, currently available anti-HBV agents have no direct effect on HBsAg transcription and protein expression. In this study, we designed a heat shock protein gp96 inhibitor p37 with the cell penetrating peptide PTD (protein transduction domain of trans-activator of transcription), which mediated p37 internalization into hepatocytes. PTD-p37 effectively suppressed HBsAg expression and viral replication both in vitro and in vivo. We further provide evidence that PTD-p37 suppressed HBV enhancer/promoter activity via p53 upregulation. Moreover, PTD-p37 had antiviral activity against a lamivudine-resistant HBV strain. Considering that suppression of HBsAg expression is a major goal for treatment of HBV infection, our results provide a basis for developing a new therapeutic approaches targeting host factors against viral expression.

Role of polycyclic aromatic hydrocarbons as a co-factor in human papillomavirus-mediated carcinogenesis.

BACKGROUND: Human papillomavirus (HPV) is an etiological agent of cervical cancer. Yet co-factors are believed to be involved in HPV-mediated carcinogenesis. Polycyclic aromatic hydrocarbons (PAHs) are considered as one of these co-factors. Epidemiologic studies have associated high PAH exposure with increased risk for cancer development. To date, many studies focus on benzo[a]pyrene, however, the role of other PAHs should not be neglected. This study aimed to compare the potential of different PAHs as a co-factor in HPV-mediated carcinogenesis, and to investigate the possible mechanisms involved. METHODS: The effect of 17 PAHs on high-risk HPV (HPV16) were examined in this study. HPV16 E7 oncogene was expressed in primary cells extracted from baby rat kidney and treated with PAHs. The co-transforming ability of PAHs were measured by colony formation index according to the number and size of transformed colonies. Effects of PAHs on proliferation of HPV-null (C33A) and -infected (CaSki) were examined using CCK-8 assay. Wound healing assay and matrigel invasion chambers were used to investigate effects of PAHs on cell motility and invasivion of HPV-null (MCF7, C33A) and -infected (SiHa) cells. RESULTS: Benzo[a]pyrene (BaP), dibenz[a,h]anthracene (DBA) and indeno[1,2,3-cd]pyrene (IDP) showed the greatest co-transforming potential in the baby rat kidney cell system. Short-term exposure to BaP, DBA, IDP and pyrene (PR) did not affect proliferation of C33A or CaSki cells, however, long-term exposure of these four PAHs led to dramatic increase in growth rate of CaSki cells by 120-140%. Besides, exposure of PAHs has an effect on cell motility and invasiveness of C33A and SiHa cells, but not for MCF7 cells. Exposure of BaP and DBA enhanced migration (1.26 to 1.40-fold) and invasion (1.68 to 1.94-fold) capacity of C33A cells. Intriguingly, exposure of all four types of PAHs boosted the migration (1.12 to 1.28-fold) and invasion (1.26 to 1.40-fold) capacity of SiHa cells. CONCLUSIONS: Our results indicate that exposure to PAHs can be a key co-factor in HPV-related cancer development. They could act on all three stages, namely initiation, promotion and progression. Further study is needed to unveil the mechanisms by which PAHs interact with HPV to cause malignancy.

T1-Mediated Nanosensor for Immunoassay Based on an Activatable MnO2 Nanoassembly.

Current magnetic relaxation switching (MRS) sensors for detection of trace targets in complex samples still suffer from limitations in terms of relatively low sensitivity and poor stability. To meet this challenge, we develop a longitudinal relaxation time (T1)-based nanosensor by using Mn2+ released from the reduction of a MnO2 nanoassembly that can induce the change of T1, and thus can greatly improve the sensitivity and overcome the "hook effect" of conventional MRS. Through the specific interaction between antigen and the antibody-functionalized MnO2 nanoassembly, the T1 signal of Mn2+ released from the nanoassembly is quantitatively determined by the antigen, which allows for highly sensitive and straightforward detection of targets. This approach broadens the applicability of magnetic biosensors and has great potential for applications in early diagnosis of disease biomarkers.

Biosynthesis of staphylococcal enterotoxin A by genetic engineering technology and determination of staphylococcal enterotoxin A in water by HPLC-ESI-TOF.

Staphylococcal enterotoxin A (SEA) was the major virulence factor of Staphylococcus aureus and a biomarker of S. aureus. To establish a fast, low cost, high accuracy, reliable, and simple method for detecting S. aureus, SEA was analyzed by HPLC-ESI-TOF. SEA was not yet commercially available in universal, so SEA was prepared before it was analyzed by HPLC-ESI-TOF. The result showed that high purified SEA was successfully prepared and SEA has normal distribution in mass spectra. A large amount of recombinant SEA (rSEA) was obtained by engineering technology and was purified by Ni affinity chromatography column, and the expression and purity of rSEA and SEA were analyzed by SDS-PAGE. The factors effected on ionization of SEA were studied, and the qualitative analysis of SEA by HPLC-ESI-TOF. The result showed that large amount of SEs expressed within a short time at 28 °C or thereabouts, and there was no impurity bands in electrophorogram after rSEA was purified by Ni affinity chromatography column. In addition, the SEA which had homologous AA sequence with wild SEA was made by rSEA. The retention of SEA in column and ionization of SEA in ESI-TOF were studied for qualitative analysis of S. aureus. The result showed that the content of formic acid in mobile phase was an important factor for ionization of SEs in ESI-TOF. And the result provided theoretical foundation for qualitative detection of S. aureus. [SEs + nH+ + mNH4+] n+m+ was shown on ESI-TOF spectra when SEA was detected by ESI-TOF in positive ion mode, and the numerical value of n+m was less than or equal to the number of basic amino acids in SEs. This method was applied to determine SEA in water samples preliminarily, and the detection limit of SEA in spiked water sample was 3 mg/kg. The limit of detection of 3 mg/kg was low sensitivity for low molecular weight matters, but it was high sensitivity for SEA which had a high molecular weight of 27 kDa. Of SEA, 3 mg/kg was equivalent to 10-4 mmol/kg of SEA. This study can provide evidence for establishing method to determine SEA in real samples.

AuGd integrated nanoprobes for optical/MRI/CT triple-modal in vivo tumor imaging.

Multi-modal imaging agents are desirable for tumor diagnosis because they can provide more information on the tumor than single-modal imaging agents. However, most reported multi-modal imaging agents are dual-modal agents rather than tri-modal agents; therefore, detailed information on the tumor may still be insufficient when such imaging agents are used. To ameliorate this issue, we synthesized a tri-modal imaging agent, composed of gold cluster and gadolinium oxide integrated nanoparticles (denoted as AuGds) using bovine serum albumin (BSA) as the template via a bio-mineralization method. The AuGds exhibit red fluorescence at ∼660 nm for optical imaging, strong X-ray absorption (around 700 HU) for CT imaging, and a high r1 value (∼12.39 mM-1 s-1) for MR imaging. After being chemically modified with folic acid (FA), the AuGds can specifically target folate receptors on KB tumor cells, and permit in vivo optical, MR, and CT imaging of xenografted tumors. By comparing these three imaging modalities, very clear structural and anatomical information on the in vivo tumor can be obtained. The AuGds show good biocompatibility, quick renal clearance, and do not induce normal tissue toxicity in vivo.

[Study of Reaction Dynamics between Bovine Serum Albumin and Folic Acid by Stopped-Flow/Fluorescence].

As a kind of coenzyme of one-carbon enzymes in vivo, folic acid belongs to B vitamins, which can interact with other vitamins and has great significance for converting among amino acids, dividing growth of cells and protein synthesis reactions. Half-life, concentration and reaction rate constant of drugs are important parameters in pharmacokinetic study. In this paper, by utilizing fluorescence spectrophotometer and stopped-flow spectrum analyzer, reaction kinetic parameters between bovine serum albumin(BSA) and folic acid in a bionic system have been investigated, which provide references for parameters of drug metabolism related to folic acid. By using Stern-Volmer equation dealing with fluorescence quenching experiments data, we concluded that under 25, 30, and 37 degrees C, the static quenching constants of folic acid to intrinsic fluorescence from bovine serum albumin were 2.455 x 10(10), 4.900 x 10(10) and 6.427 x 10(10) L x mol(-1) x s(-1) respectively; The results of kinetic reaction rate have shown that the reaction rate of BSA and folic acid are greater than 100 mol x L(-1) x s(-1) at different temperatures, pH and buffering media, illustrating that the quenching mechanism between BSA and folic acid is to form composite static quenching process. Reaction concentration of bovine serum albumin and its initial concentration were equal to the secondary reaction formula, and the correlation coefficient was 0.998 7, while the half-life (t1/2) was 0.059 s at physiological temperature. With the increase of folic acid concentration, the apparent rate constant of this reaction had a linear increasing trend, the BSA fluorescence quenching rate constant catalyzed by folic acid was 3.174 x 10(5) mol x L(-1) x s(-1). Furthermore, with different buffer, the apparent rate constant and reaction rate constant of BSA interacting with folic acid were detected to explore the influence on the reaction under physiological medium, which is of great significance to determine the clinical regimen, forecast the efficacy and toxicity of drugs and rational drug.

[Research on the Reaction Dynamics between Peroxynitrite and Tyrosine Catalyzed with Mimic Enzyme through Flow Injection Analyzer].

As a highly active free radical in vivo, peroxynitrite can damage various biological macromolecules and cause a series of major diseases, which is of great significance to determine its content and reactive mechanism. It is very difficult to capture the dynamic process of peroxynitrite due to its active property and fast reacted rate. In this paper, we firstly explored the kinetic characteristics of peroxynitrite and tyrosine with the presence of Hemoglobin and Hemin by using flow injection analyzer. The results showed that the oxidation processes of peroxynitrite and tyrosine catalyzed with hemoglobin and hemin were in accordance with Michaelis-Menten's dynamics law; Based on the Michaelis constant (K(m)) and the maximum initial rate (V(max)), we deduced the reaction mechanism that peroxynitrite, catalyzed by mimic enzymes, directly oxidized tyrosine combined with mimic enzymes, rather than decomposed to · OH and O(-2)·; In addition, we detected the rate constant of the reaction catalyzed by these two kinds of enzymes at different temperature and pH, resulting that the optimum conditions of hemoglobin to catalyze this system were 25 ℃ and pH 8, the rate constant was 1.035×10(6) mol·L(-1) · s(-1), while hemin was 37 ℃ and pH 9.5, the rate constant was 6.842×10(5) mol·L(-1) · s(-1); Comparing the kinetic parameters, K(m)Hb(4.46 µmol·L(-1))V(Hemin)(max)(0.026 ΔIF/s), we discovered that the rate constant of hemoglobin in optimum condition was greater than that in hemin, and the catalytic activity of hemoglobin was higher than that of hemin. All these results have provided kinetic parameters for the study of determining the content of peroxynitrite and exploring its reaction mechanism with enzymatic method. It also laid a theoretical foundation for developing new diagnosis technology to prevent and cure diseases caused by free radicals in vivo.

Peroxynitrite induced fibrinogen site identification.

An increasing number of peroxynitrite-mediated fibrinogen nitrifications have been associated with thrombotic diseases. However, few reports related to priority nitrified fibrinogen injury sites exist. In this paper, an improved method, which simulated the environment in vivo, was used to inspect the structural changes of fibrinogen treated with peroxynitrite and LC-MS/MS in order to investigate the fibrinogen injury sites. The SDS-PAGE results indicated that γ subunits of Fg were vulnerable to oxidative/nitrative modifications induced by peroxynitrite. An in-depth analysis of fibrinogen γ chain (Fg I) nitration site identification and susceptibility to peroxynitrite-utilizing LC-MS/MS strategy was performed. Based on a large dataset, the results indicated the priority injury sites during database searching were Y96, Y262, Y274, Y348, and Y363; these results could be applied to biomedical studies.

Metabolic transformation of indeno[1,2,3-cd]pyrene and toxicities of its metabolites.

A porphyrin/peroxynitrite biomimetic system was used to study the metabolism of indeno[1,2,3-cd]pyrene (IND) induced by peroxynitrite. The metabolites were identified using high-performance liquid chromatography coupled with electro-spray ionization tandem mass spectrometry as OH-IND, IND-quinone and 2NO2-IND. By stopping the reaction at different stages, we discovered that IND was first transformed to IND-quinone and 2NO2-IND, which were then transformed to OH-IND. Mutation assays including Ames tests and cell transformation experiments showed enhancement of the mutagenicity after the activation by the peroxynitrite/Fe(III)porphyrin system. The results also showed that 2NO2-IND and IND-quinone played key roles in the mutagenicity of PAHs after metabolic activation.

[Determination of five ß2-agonist residues in pork by ultra-high performanc liquid chromatography-tandem mass spectrometry with molecularly imprinted solid phase extraction].

An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with molecularly imprinted solid phase extraction for the determination of five ß2-gonists residues in pork has been developed. After the sample preparation, the ammonium acetate/acetic acid buffer was added, followed by the internal standard and ß-glucuronidase/arylsulfatase enzyme. The solution was incubated at 55 °C for 2 h. After adjusting the pH of the solution, it was purified by a molecularly imprinted solid phase extraction column, then analyzed on a BEH C18 column with methanol-0.1% (v/v) formic acid aqueous solution as the mobile phases in gradient elution mode. The MS/MS analysis was in positive ion mode and multiple reaction monitoring mode. The analytes were quantified by the internal standard method. The limits of detection (LODs, S/N = 3) and the limits of quantification (LOQs, S/N = 10) were 0.005-0.009 µg/kg and 0.015-0.025 µg/kg, respectively. In the range of 0-10 µg/kg, the correlation coefficients of linear calibration curves were not less than 0.993 3. At the spiked levels of 0.25, 1.0 and 5.0 µg/kg, the recoveries were 80.4%-92.9% with the relative standard deviations of 1.3%-6.3%. The method is of high sensitivity, good reproducibility, high recovery, and is useful for the simultaneous determination of multiple ß2-agonists residues.

Effects of Mn (II) on peroxynitrite nitrifying fibrinogen.

Fibrinogen is a plasma glycoprotein that is an established cardiovascular risk and it participates in the blood-clotting mechanism. Nitrated fibrinogen has been shown to inhibit platelet aggregation and thrombus formation. However, there are only a few reports relating to the activity and structural changes of nitrified fibrinogen when metal ions are present in the reaction. Mn (II) ion plays an important physiological role in the nervous system and cardiac function. In this study, we use UV-Vis, 3D-fluorescence, SDS-PAGE electrophoresis and Von-Clauss to detect 3-nitrotyrosine (3-NT) production and the activity changes of fibrinogen after nitration and oxidation damage caused by ONOO- in the presence of Mn (II). Results showed that Mn (II) can enhance the production of 3-NT in fibrinogen, promote fluorescence quenching of fibrinogen, and increase the injury to γ and Aα chains of fibrinogen in the presence of peroxynitrite. Consequently, Mn (II) promotes concentration dependent fibrinogen nitrification damage and significantly reduces the biological activity of nitrified fibrinogen.

Identification of nitration sites by peroxynitrite on p16 protein.

Injury of p16 has been implicated in some cancers. In this paper, we focus on the need for identification of peroxynitrite-dependent nitration sites on p16 with HPLC-MS/MS method. Two mono-nitrated residues Tyr129 and Tyr44 were detected in the course of p16 modification induced by peroxynitrite at relative low doses. As suggested by peptide mapping sequence analysis, Tyr44 was more liable to be nitrated by ONOO(-). Study on the chemical environment of two Tyr residues reveals that steric hindrance may be the structural determinant for the nitration sequence. Through technique of SDS-PAGE, ONOO(-) could induce p16 nitration, even strongly damage the combination of p16 with CDK4, which further influence p16's activity.