Search

Krebs ist nach wie vor eine der häufigsten Todesursachen weltweit und eine Vielzahl an Krebsarten ist aktuell nicht heilbar (Sung et al., 2021). Trotz der Entdeckung hochwirksamer Pt-basierter Tumortherapeutika wie Cisplatin, Oxaliplatin und Carboplatin ist die Entwicklung metallbasierter Krebsmedikamente mit günstigerem Nebenwirkungsprofil und verminderter Resistenzentwicklung notwendig. Neben Pt-Verbindungen werden auch Ru-, Os-, Rh-, Ir-, Ti-, Au-Komplexe intensiv als Antitumormittel untersucht. Viele Vertreter dieser Substanzklassen scheinen ihre Wirkung nicht primär über Bindung der DNA zu entfalten, wie es bei Cisplatin der Fall ist, sondern durch Interaktion mit Proteinen (Dyson and Sava, 2006). Das Verständnis molekularer Interaktionen von experimentellen Metallkomplexen mit biologischen Strukturen ist entscheidend für die Entwicklung neuer Chemotherapeutika. Besonders massenspektrometrische Verfahren werden zur Charakterisierung molekularer Interaktionen und zur Identifizierung biologischer Targets eingesetzt (Hartinger et al., 2013). In dieser Arbeit wurden 16 ausgewählte Metallkomplexe und ihre molekularen Interaktionen mit verschiedenen Proteinen mittels HPLC-ESI-MS analysiert. Die hierbei gewonnenen Daten liefern wertvolle Erkentnisse zur Ausbildung und Kinetik gebildeter Addukte und können einen Beitrag zur Entwicklung neuer therapeutischer Metallkomplexe leisten. Ziel dieser Arbeit war die Entwicklung einer HPLC-ESI-MS(MS2)-Methode zur Bestimmung von Bindungspräferenzen der Testsubstanzen, welche erstmalig eine simultane Messung von Proteinen und Oligonukleotiden im positiven Modus ermöglicht. Als Oligonukleotid wurde ein 8-mer mit einer internen GG-Sequenz eingesetzt, welche als bekannte Bindungsstelle zu Cisplatin gilt. Durch HCD-Fragmentierung von [8-mer + Pt(NH3)2]-Addukten und anschließender Analyse des erhaltenen Fragmentspektrums mit der Aom2S-Software wurde die Bindung zu dieser Sequenz überprüft. Bei der Methodenentwicklung war die Untersuchu, Cancer remains one of the leading causes of death worldwide and many cancer-types are currently incurable (Sung et al., 2021). Despite the discovery of highly effective Pt-based anticancer drugs such as cisplatin, oxaliplatin and carboplatin, there is a need to develop metal-based anticancer drugs with a more favourable side-effect profile and lower resistance development. In addition to Pt compounds, Ru, Os, Rh, Ir, Ti and Au complexes are also being intensively investigated as antitumour agents. Many representatives of these substance classes do not appear to exert their effect primarily by binding DNA, as is the case with cisplatin, but rather by interacting with proteins (Dyson and Sava, 2006). Understanding the molecular interactions of experimental metal complexes with biological structures is crucial for the development of new chemotherapeutics. Mass spectrometric methods in particular are used to characterize molecular interactions and identify biological targets (Hartinger et al., 2013). In this study, 16 selected metal complexes and their molecular interactions with various proteins were analyzed using HPLC-ESI-MS. The data obtained provide valuable insights into the formation and kinetics of formed adducts and can contribute to the development of new metallodrugs. The aim of this thesis was to determine the binding preferences of the tested metal complexes by developing an HPLC-ESI-MS(MS2) method, which enables for the first time the simultaneous measurement of proteins and oligonucleotides in positive ion mode. The oligonucleotide used was an 8-mer with an internal GG-sequence, which is a known binding site for cisplatin. The ability for cisplatin to bind to this sequence was verified by HCD fragmentation of [8-mer + Pt(NH3)2] adducts and subsequent analysis of the resulting fragment spectrum using the Aom2S software. During method development, the analysis of oligonucleotides in positive ion mode in the presence of other biomolecules was associated with, eingereicht von: Slaviero Daniel, Im Titel ist 2 hochgestellt, in englischer Sprache, Masterarbeit Universität Innsbruck 2023

V teoretické části jsou popsány obecné vlastnosti, přípravy a strukturní motivy amidů kovů 1. a 14. skupiny periodického systému a jejich využití v transmetalačních reakcích se zaměřením na amidy kovů obsahující bis(amido)silylový ligand. V experimentální části je popsána příprava nových bis(amido)silylových ligandů (I - III), následná syntéza cílených stannylenů (X, XI) a charakterizace všech těchto sloučenin pomocí multinukleární NMR spektroskopie a rentgenové difrační analýzy monokrystalického materiálu. Dále bylo připraveno 6 aduktů protoligandů s n-BuLi a MeLi (IV - IX), které byly následně charakterizovány pomocí multinukleární NMR spektroskopie a ve většině případů i za využití retgenové difrakční analýzy. Také je zde popsána syntéza a charakterizace trianionického trilithného komplexu [Ph(Me)Si(Ar*NLi)2]Li, který byl připraven z odpovídajícího aduktu protoligandu s n-BuLi. Mechanismus této reakce byl zkoumán pomocí DFT kalkulací., In the theoretical part, the general properties, preparations, structural motifs of metal amides of Groups 1 and 14 of the Periodic Table, are described, with emphasis on metal amides bearing bis(amido)silyl ligand. Also use of bis(amido)silyl amides in transmetalations and other types of reactions is described there. In the experimental part, the preparation of new bis(amido)silyl ligands (I-III), subsequent synthesis of targeted stannylenes (X, XI), and characterization of all these compounds using multinuclear NMR spectroscopy and X-ray diffraction analysis of grown single crystals are described. Additionally, 6 adducts of protoligands with n-BuLi and MeLi, which were subsequently characterized using multinuclear NMR spectroscopy and in most cases also by X-ray diffraction analysis of single crystals. The synthesis and characterization of trianionic trilithium complex [Ph(Me)Si(Ar*NLi)2]Li, which was prepared from the corresponding aduct of protoligand with n-BuLi, is also described. The mechanism of this reaction was investigated using DFT calculations., Fakulta chemicko-technologická, Diplomant seznámil komisi se svojí diplomovou prací. Dále reagoval na připomínky oponenta. Zodpověděl otázky členů komise: Vysvětlete symboly prostorových grup použitých v diplomové práci. Diskutujte výpočty u studovaných sloučenin., Dokončená práce s úspěšnou obhajobou

Hemoglobin and serum albumin, two prevalent proteins in human blood, contain unbound cysteine thiol moieties, creating a nucleophilic site with the potential for covalent modification by reactive chemical species. These covalent modifications, called “adducts”, are stable entities that accumulate during acute and chronic exposure and remain covalently bound for the life-span of the protein. Despite their current use as exposure markers for a variety of compounds, the use of adducts in assessing exposure to drugs of abuse has not yet been explored. The goal of this work was to examine the in vitro adduct forming capability of selected drugs of abuse with hemoglobin to provide additional proof of principle for the development of a real-world detection and monitoring analysis method. This goal was accomplished by first analyzing the binding capabilities of the drugs of interest with glutathione, a smaller tripeptide. Use of protein adducts as biomarkers of drug exposure may allow for an increased window of detection, from several days to several months, as compared to current blood analysis methods. In total, there were 16 drugs analyzed in the research, and they covered a wide range of abused drugs, including cocaine, methamphetamine, and Δ 9 -THC. Results from the glutathione trials showed that 10 of the 16 the drugs of interest were able to form covalent adducts with the free thiol moiety, with four drugs forming more than one novel adduct. The MS results for hemoglobin showed 11 adducts formed for five of the drugs under investigation. Additional MS/MS confirmatory data was obtained for two of those 11 adducts. I successfully identifyied adducts formed between drugs of abuse and glutathione and hemoglobin, which have the potential to be used as long-term biomarkers of exposure.

4-hydroxy-2-nonenal (HNE), a major non-saturated aldehyde product of lipid peroxidation, has been extensively studied as a signaling messenger. In these studies a wide range of HNE concentrations have been used, ranging from the unstressed plasma concentration to far beyond what would be found in actual pathophysiological condition. In addition, accumulating evidence suggest that signaling protein modification by HNE is specific with only those proteins with cysteine, histidine, and lysine residues located in certain sequence or environments adducted by HNE. HNE-signaling is further regulated through the turnover of HNE-signaling protein adducts through proteolytic process that involve proteasomes, lysosomes and autophagy. This review discusses the HNE concentrations and exposure modes used in signaling studies, the selectivity of the HNE-adduction site, and the turnover of signaling protein adducts.

4-hydroxy-2-nonenal (HNE), a major non-saturated aldehyde product of lipid peroxidation, has been extensively studied as a signaling messenger. In these studies a wide range of HNE concentrations have been used, ranging from the unstressed plasma concentration to far beyond what would be found in actual pathophysiological condition. In addition, accumulating evidence suggest that signaling protein modification by HNE is specific with only those proteins with cysteine, histidine, and lysine residues located in certain sequence or environments adducted by HNE. HNE-signaling is further regulated through the turnover of HNE-signaling protein adducts through proteolytic process that involve proteasomes, lysosomes and autophagy. This review discusses the HNE concentrations and exposure modes used in signaling studies, the selectivity of the HNE-adduction site, and the turnover of signaling protein adducts.

4-hydroxy-2-nonenal (HNE), a major non-saturated aldehyde product of lipid peroxidation, has been extensively studied as a signaling messenger. In these studies a wide range of HNE concentrations have been used, ranging from the unstressed plasma concentration to far beyond what would be found in actual pathophysiological condition. In addition, accumulating evidence suggest that signaling protein modification by HNE is specific with only those proteins with cysteine, histidine, and lysine residues located in certain sequence or environments adducted by HNE. HNE-signaling is further regulated through the turnover of HNE-signaling protein adducts through proteolytic process that involve proteasomes, lysosomes and autophagy. This review discusses the HNE concentrations and exposure modes used in signaling studies, the selectivity of the HNE-adduction site, and the turnover of signaling protein adducts.

This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation. The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established. In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be >400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation. An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment., At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation. The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established. In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be >400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation. An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment., At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation. The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established. In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be >400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation. An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment., At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation. The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established. In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be >400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation. An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment., At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation. The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established. In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be >400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation. An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment., At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation. The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established. In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be >400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation. An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment., At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Aromatic hydrocarbons are ubiquitous pollutants. Many of the hydrocarbons such as benzene and benzo[a]pyrene are genotoxic and are known risk factors for cancers. The genotoxicity comes from the metabolic activation of the parent compound to a reactive electrophilic form, which binds to the nucleophilic DNA and albumin to form stable adducts. Because adduct represents the biologically effective dose and albumin has relatively long half-life (~ 1 month) and is abundant in blood (~45 mg/ml), albumin adduct becomes a promising candidate to assess exposure to aromatic hydrocarbons in cancer epidemiology. This dissertation can be roughly divided into two parts. In the first part, an antibody was used to quantitatively assess benzo[a]pyrene exposure. A sandwich enzyme-linked immunosorbent assay (ELISA), using a commercially available antibody, was developed to measure benzo[a]pyrene diol epoxide - albumin adduct (BPDE-HSA). The assay is much simpler and faster to use and is 10 times more sensitive than the traditional competitive ELISA to detect BPDE-HSA. The method was further revised to be directly compatible with plasma, hence eliminated all the tedious HSA isolation and purification steps. Finally, the revised assay was validated with two different sets of archived plasma samples. In the second part, another antibody was used in an enrichment step for the albumin adductomics. To characterize albumin adducts with liquid chromatography - mass spectrometry, they have to be digested and detected as peptide adducts. Cysteine (Cys34) is a known nucleophilic hotspot on the albumin that scavenges reactive aromatic electrophiles in blood (such as benzoquinone and naphthoquinones), therefore a semi-targeting antibody was designed to capture all peptides with electrophiles bound on the Cys34 (T3 adducts). After incorporating the antibody enrichment step into the adductomic workflow, results shown that the antibody enrichment has huge potential to increase the detection rate of l

Aromatic hydrocarbons are ubiquitous pollutants. Many of the hydrocarbons such as benzene and benzo[a]pyrene are genotoxic and are known risk factors for cancers. The genotoxicity comes from the metabolic activation of the parent compound to a reactive electrophilic form, which binds to the nucleophilic DNA and albumin to form stable adducts. Because adduct represents the biologically effective dose and albumin has relatively long half-life (~ 1 month) and is abundant in blood (~45 mg/ml), albumin adduct becomes a promising candidate to assess exposure to aromatic hydrocarbons in cancer epidemiology. This dissertation can be roughly divided into two parts. In the first part, an antibody was used to quantitatively assess benzo[a]pyrene exposure. A sandwich enzyme-linked immunosorbent assay (ELISA), using a commercially available antibody, was developed to measure benzo[a]pyrene diol epoxide - albumin adduct (BPDE-HSA). The assay is much simpler and faster to use and is 10 times more sensitive than the traditional competitive ELISA to detect BPDE-HSA. The method was further revised to be directly compatible with plasma, hence eliminated all the tedious HSA isolation and purification steps. Finally, the revised assay was validated with two different sets of archived plasma samples. In the second part, another antibody was used in an enrichment step for the albumin adductomics. To characterize albumin adducts with liquid chromatography - mass spectrometry, they have to be digested and detected as peptide adducts. Cysteine (Cys34) is a known nucleophilic hotspot on the albumin that scavenges reactive aromatic electrophiles in blood (such as benzoquinone and naphthoquinones), therefore a semi-targeting antibody was designed to capture all peptides with electrophiles bound on the Cys34 (T3 adducts). After incorporating the antibody enrichment step into the adductomic workflow, results shown that the antibody enrichment has huge potential to increase the detection rate of l

As organisms age, a considerable decrease in protein synthesis takes place in all tissues. Among the possible causes of the decline of translation in old animals are the modifications of elongation factor-2 (eEF- 2). eEF-2 occupies an essential role in protein synthesis where it catalyzes the ribosomal translocation reaction. eEF-2 is particularly sensitive to increased oxidative stress. However, all oxidants do not affect eEF-2, only compounds that increase lipid peroxidation. As peroxides are unstable compounds, they decompose and generate a series of highly reactive compounds, including aldehydes malondialdehyde (MDA) and 4-hydroxynoenal (HNE). We have previously reported that hepatic eEF-2 forms adducts with low-molecular weight aldehydes, MDA and HNE. Therefore, the protection of eEF-2 must be specifically carried out by a compound with lipoperoxyl radicalscavenging features such as melatonin. In this article, we show the ability of melatonin to protect against the changes that occur in the eEF-2 under conditions of lipid peroxidation induced by cumene hydroperoxide (CH), a compound used experimentally to induce lipid breakdown. As experimental models, we used cultured cells and rats treated with this oxidant compound. eEF-2 levels, adduct formation of this protein with MDA and HNE, and lipid peroxides were determined. In the cultured cells, protein synthesis rate was also measured. Our results show that melatonin prevented the molecular changes in eEF-2 and the decline in protein synthesis rate secondary to lipid peroxidation. The results also show that serum levels of several hormones were affected by CH-induced oxidative stress, which was partially or totally prevented by melatonin.

As organisms age, a considerable decrease in protein synthesis takes place in all tissues. Among the possible causes of the decline of translation in old animals are the modifications of elongation factor-2 (eEF- 2). eEF-2 occupies an essential role in protein synthesis where it catalyzes the ribosomal translocation reaction. eEF-2 is particularly sensitive to increased oxidative stress. However, all oxidants do not affect eEF-2, only compounds that increase lipid peroxidation. As peroxides are unstable compounds, they decompose and generate a series of highly reactive compounds, including aldehydes malondialdehyde (MDA) and 4-hydroxynoenal (HNE). We have previously reported that hepatic eEF-2 forms adducts with low-molecular weight aldehydes, MDA and HNE. Therefore, the protection of eEF-2 must be specifically carried out by a compound with lipoperoxyl radicalscavenging features such as melatonin. In this article, we show the ability of melatonin to protect against the changes that occur in the eEF-2 under conditions of lipid peroxidation induced by cumene hydroperoxide (CH), a compound used experimentally to induce lipid breakdown. As experimental models, we used cultured cells and rats treated with this oxidant compound. eEF-2 levels, adduct formation of this protein with MDA and HNE, and lipid peroxides were determined. In the cultured cells, protein synthesis rate was also measured. Our results show that melatonin prevented the molecular changes in eEF-2 and the decline in protein synthesis rate secondary to lipid peroxidation. The results also show that serum levels of several hormones were affected by CH-induced oxidative stress, which was partially or totally prevented by melatonin.

The kinetics and thermodynamics of ester hydrolysis were studied experimentally in a laboratory-scale batch reactor by using ethyl formate as the model molecule. The effects of the reaction conditions, such as temperature, excess water, complexing agent and initial acid charge upon the ester hydrolysis process were investigated and a kinetic model was developed for the system. Autocatalytic kinetics was observed experimentally, which was due to the carboxylic acid formed during the reaction. The reaction rate was further enhanced and the equilibrium was shifted to the product side by adding a complexing agent into the reaction mixture. A mathematical model comprising the mass balances and rate equations were developed for the system by assuming quasi-equilibrium hypothesis for the reaction involving the complexing agent. A robust calculation scheme was developed for the estimation of the kinetic and thermodynamic parameters from experimental data. The proposed model was able to predict the experimental results satisfactorily.

Allergic contact dermatitis (ACD) is a common T cell mediated skin disease. Small reactive organic molecules called haptens induce ACD. Haptens are not recognized by themselves, but need to bond to endogenous proteins in the skin, and processed further into complete antigens. Antigen specific T cells then recognize the resulting antigen. Although the haptens are well known, the chemical reactions of haptens inducing ACD is not well known. Neither are the chemical structures of the complete antigens associated with ACD in humans. This study is composed of three parts. Reactivity study of strong sensitizers like 2,4-dinitrofluorobenzene (DNFB), 1,4-benzoquinone (BQ), 4-t-butyl-1,2-benzoquinone (tBuBQ) and cis-1,2-hexahydrophthalic-anhydride (HHPA) under physiologic conditions with nucleophilic amino acids and peptides by analytical methods (HPLC, LC/MS/MS). The new reaction products were isolated and their structures were determined by NMR and MS. We found that most haptens reacted predominantly with cystein and cystein containing peptides. We then synthesized an array of 2,4-dinitrophenyl- (Dnp) modified collagen II peptides, as complete antigens, by solid phase peptide synthesis (SPPS). In a MHC class II (H2-Aq) restricted T cell model of ACD, we evaluated the responses of these antigens. The length of the amino acid side chain bonding the Dnp-group determined the T cell response. Substitution of lysine for ornithine gave a complete loss of T cell recognition. We finally used homology modeling to construct a model of the H2-Aq/antigen/TCR complex, a part of the ”immunological synapse”. Our new model could explain the experimental T cell responses in terms of a defined structure of the MHC/antigen, and how this structure is displayed for the TCR. This study may contribute to better diagnosis, and possibly a cure of ACD in the future.

The effect of cytochrome P4501A (CYP1A)induction on cell-specific benzo[a]pyrene (BaP) adduct formation was studied in rainbow trout (Oncorhynchus mykiss) gills. Fish preexposed to β-naphthoflavone (βNF) or caged in a polluted river were exposed to waterborne 3H-benzo[a]pyrene (3H-BaP). The 3H-benzo[a]pyrene adducts in the gill filaments were localized by autoradiography and CYP1A protein by immunohistochemistry. Ethoxyresorufin O-deethylase (EROD) activity was measured using a gill filament-based ex vivo assay. Branchial 3H-BaP binding and EROD activity were enhanced by exposure to βNF or to the river water, and completely blocked by the CYP1A inhibitor ellipticine. The predominant sites of adduct formation were in epithelium of the secondary lamellae and in epithelium of the efferent edge of the gill filament. In βNF-exposed fish, the strongest CYP1A immunoreactivity was observed in differentiating cells and in pillar cells. In fish caged in the polluted river, strong CYP1A immunoreactivity was found in most cells in the secondary lamellae, whereas the primary lamellae were almost devoid of immunoreactivity. Our results reveal a discrepancy between the localization of CYP1A protein and BaP adducts in the gill. Consequently, other factors, such as bioavailability of waterborne polycyclic aromatic hydrocarbons (PAHs) to the target cells, are important for the localization of PAH adducts in the gill.

Pseudo-molecular ion formation is an artifact common to most analyses performed by electrospray ionization mass spectrometry. These species are non-covalent complexes formed between an analyte of interest and any other components (such as mobile phase, additives, and impurities) present in the ionized sample band. Published literature addresses pseudo-molecular ion formation in routine analyses as well as in complicated molecular recognition processes. The majority of these works are directed towards the formation of complexes in the positive ionization mode. Consequently, investigation of pseudo-molecular ion formation in the negative ionization mode is a logical extension of work in this area. Experiments presented here detail the work performed on elucidation of factors controlling ionization efficiency of aromatic acid pseudo-molecular ions by electrospray ionization in the negative ionization mode. Sets of tested acidic analytes, including ibuprofen derivatives and benzoic acid derivatives, were analyzed in the presence of various solution systems by flow injection analysis to determine the effect of pH, concentration, injection volume, and instrumental parameters on dominant ion forms observed in the mass spectra. These ion forms correspond to a deprotonated molecular ion ([M-H]-), a hydrogen-bound dimer ion ([2M-H]-), and a sodium-bridged dimer ion ([2M-2H+Na]-). Report of the latter ion form is unique to this work. Response of these ion forms were found to vary greatly with changing solution parameters, particularly in the presence of common LC-MS modifiers, such as triethylamine, acetic acid, formic acid, and ammonium formate. Results point to the formation of the sodium-bridged dimer ion during gas-phase processes following the release of ions from disintegrated droplets. Ab initio theoretical calculations and correlations with calculated solution phenomena (such as pKa and log P) were used to elucidate structural arrangements and dominant factors controll

The last decade has seen considerable attention focused on the formation of suitable precursors to III-V compound semiconductors. As a part our research in this area, we recently reported the isolation of 'mixed bridge' compounds of the heavier group III elements (i.e., four-membered ring systems whose chlorine atom should allow introduction of other functional groups within these systems. From these precursors, the isolation of mixed III-V compound semiconductors may be possible. Herein we report the reactions of several mixed bridge compounds. All manipulations were performed using general Schlenk, dry box, or high vacuum techniques. Solvent (including those for NMR spectra) were appropriately dried and distilled under argon prior to use.

The 2:1 mole ratio reaction of InCl3 and P(SiMe3)3 in a toluene/THF mixture was investigated. Reaction appears to occur in a 1: 1 mole fashion, since the products obtained are unreacted InCl3 (48.5% recovered) as InCl3.2THF and a fine powder which roughly analyzes to be Cl2In3P3(SiMe3)2)n. The latter was characterized by melting point and partial elemental analysis. The X-ray crystal structure of InCl3.2THF is reported. InCl3.2THF crystallizes in the orthorhombic space group Pbcn (D2h14), No.60) with unit cell parameters of a = 10.802(1) A, b = 10.101(1) A, c = 13.083(1) A, v = 1427.5(4) A3, and Dcalcd = 1. 700 g cm-3 for Z = 4 R 0.042 (Rw = 0.058) and is an example of a neutral pentacoordinated bis-adduct of an indium(III) trihalide. The crystal structure consists of discrete monomeric units with no abnormally short intermolecular separations. The geometry about the indium atom approximates to trigonal bipyramidal with oxygen atoms of each of the THF molecules occupying axial positions In-0 = 2.257(9) A, 0-In-0 = 178.5(3) deg while the three chlorine atoms (mean In-Cl = 2.321 A) lie in the equatorial plane.

The adduct derived from the reaction of 1-[(4'-methoxyphenylimino)-methyl]-2-naphthol with triphenyltin(IV) chloride, and which exhibits 1 : 1 stoichiometry (ligand: organotin), has been characterized spectroscopically and by an X-ray structural analysis. The complex possesses trigonal bipyramidal geometry around the Sn atom with the three phenyl rings in equatorial positions; coordination of the ligand to the metal occurs via the phenolic oxygen atom.

Since our introduction in 1986 of the use of the silyl halide elimination reaction for forming the Gallium Arsenide bond, we have found it to be a versatile tool for the synthesis of interesting compounds. Using reagents of the general form RnAs(SiMe3)3-n and R'nGaX3-n (n = 0, 1, 2), a number of monomeric, dimeric, trimeric, and mixed bridge arsinogallanes have been prepared. However, some of the most basic chemical reactions in this series, viz. those between As(SiMe3)3 and GaX3, remained largely unexplored. Previously, we reported that reaction of GaX3 (X = Chlorine, Bromine) and As(SiMe3)3 in a 1:1 mole ratio yields products which, upon being heated with a cool flame, give microcrystalline GaAs. Others have shown that GaAs made in this way consists of particles small enough to be expected to exhibit quantum confinement effects.