Markers of genetic susceptibility in human environmental hygiene and toxicology : the role of selected CYP, NAT and GST genes

Inherited genetic traits co-determine the susceptibility of an individual to a toxic chemical. Special emphasis has been put on individual responses to environmental and industrial carcinogens, but other chronic diseases are of increasing interest. Polymorphisms of relevant xenobiotic metabolising enzymes may be used as toxicological susceptibility markers. A growing number of genes encoding enzymes involved in biotransformation of toxicants and in cellular defence against toxicant-induced damage to the cells has been identified and cloned, leading to increased knowledge of allelic variants of genes and genetic defects that may result in a differential susceptibility toward environmental toxicants. "Low penetrating" polymorphisms in metabolism genes tend to be much more common in the population than allelic variants of "high penetrating" cancer genes, and are therefore of considerable importance from a public health point of view. Positive associations between cancer and CYP1A1 alleles, in particular the *2C I462V allele, were found for tissues following the aerodigestive tract. Again, in most cases, the effect of the variant CYP1A1 allele becomes apparent or clearer in connection with the GSTM1 null allele. The CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squameous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has also pointed to interactive effects. Of particular interest for the industrial and environmental field is the isozyme CYP2E1. Several genotypes of this isozyme have been characterised which seem to be associated with different levels of expression of enzyme activity. The acetylator status for NAT2 can be determined by genotyping or by phenotyping. In the pathogenesis of human bladder cancer due to occupational exposure to "classical" aromatic amines (benzidine, 4-aminodiphenyl, 1-naphthylamine) acetylation by NAT2 is regarded as a detoxication step. Interestingly, the underlying European findings of a higher susceptibility of slow acetylators towards aromatic amines are in contrast to findings in Chinese workers occupationally exposed to aromatic amines which points to different mechanisms of susceptibility between European and Chinese populations. Regarding human bladder cancer, the hypothesis has been put forward that genetic polymorphism of GSTM1 might be linked with the occurrence of this tumour type. This supports the hypothesis that exposure to PAH might causally be involved in urothelial cancers. The human polymorphic GST catalysing conjugation of halomethanes, dihalomethanes, ethylene oxide and a number of other industrial compounds could be characterised as a class theta enzyme (GSTT1) by means of molecular biology. "Conjugator" and "non-conjugator" phenotypes are coincident with the presence and absence of the GSTT1 gene. There are wide variations in the frequencies of GSTT1 deletion (GSTT1 *0/0) among different ethnicities. Human phenotyping is facilitated by the GST activity towards methyl bromide or ethylene oxide in erythrocytes which is representative of the metabolic GSTT1 competence of the entire organism. Inter-individual variations in xenobiotic metabolism capacities may be due to polymorphisms of the genes coding for the enzymes themselves or of the genes coding for the receptors or transcription factors which regulate the expression of the enzymes. Also, polymorphisms in several regions of genes may cause altered ligand affinity, transactivation activity or expression levels of the receptor subsequently influencing the expression of the downstream target genes. Studies of individual susceptibility to toxicants and gene-environment interaction are now emerging as an important component of molecular epidemiology.

Semisynthetic aminoglycoside antibiotics : toward biomimetic synthesis, evasion of bacterial resistance and reduced toxicity

Les antibiotiques aminoglycosidiques sont des agents bactéricides de grande valeur et d’efficacité à large spectre contre les pathogènes Gram-positifs et Gram-négatifs, dont plusieurs membres naturels et semisynthétiques sont importants dans l’histoire clinique depuis 1950. Des travaux crystallographiques sur le ribosome, récompensés par le prix Nobel, ont démontré comment leurs diverses structures polyaminées sont adaptées pour cibler une hélice d’ARN dans le centre de codage de la sous-unité 30S du ribosome bactérien. Leur interférence avec l’affinité et la cinétique des étapes de sélection et vérification des tARN induit la synthèse de protéines à basse fidélité, et l’inhibition de la translocation, établissant un cercle vicieux d’accumulation d’antibiotique et de stress sur la membrane. En réponse à ces pressions, les pathogènes bactériens ont évolué et disséminé une panoplie de mécanismes de résistance enzymatiques et d’expulsion : tels que les N acétyltransférases, les O phosphotransférases et les O nucleotidyltransférases qui ciblent les groupements hydroxyle et amino sur le coeur des aminoglycosides; des méthyl-transférases, qui ciblent le site de liaison ribosomale; et des pompes d’expulsion actives pour l’élimination sélective des aminoglycosides, qui sont utilisés par les souches Gram-négatives. Les pathogènes les plus problématiques, qui présentent aujourd’hui une forte résilience envers la majorité des classes d’antibiotiques sur le bord de la pan-résistance ont été nommés des bactéries ESKAPE, une mnémonique pour Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa et Enterobacteriaceae. La distribution globale des souches avec des mécanismes de résistance envers les standards cliniques aminoglycosides, tels que la tobramycine, l’amikacine et la gentamicine, est comprise entre 20 et 60% des isolées cliniques. Ainsi, les aminoglycosides du type 4,6-disubstitués-2-deoxystreptamine sont inadéquats comme thérapies anti-infectieuses à large spectre. Cependant, la famille des aminoglycosides 4,5-disubstitués, incluant la butirosine, la neomycine et la paromomycine, dont la structure plus complexe, pourrait constituter une alternative. Des collègues dans le groupe Hanessian et collaborateurs d’Achaogen Inc. ont démontré que certains analogues de la paraomomycine et neomycine, modifiés par désoxygénation sur les positions 3’ et 4’, et par substitution avec la chaîne N1-α-hydroxy-γ-aminobutyramide (HABA) provenant de la butirosine, pourrait produire des antibiotiques très prometteurs. Le Chapitre 4 de cette dissertation présente la conception et le développement d’une stratégie semi-synthétique pour produire des nouveaux aminoglycosides améliorés du type 4,5 disubstitués, inspiré par des modifications biosynthétiques de la sisomicine, qui frustrent les mécanismes de résistance bactérienne distribuées globalement. Cette voie de synthèse dépend d’une réaction d’hydrogénolyse de type Tsuji catalysée par palladium, d’abord développée sur des modèles monosaccharides puis subséquemment appliquée pour générer un ensemble d’aminoglycosides hybrides entre la neomycine et la sisomicine. Les études structure-activité des divers analogues de cette nouvelle classe ont été évaluées sur une gamme de 26 souches bactériennes exprimant des mécanismes de résistance enzymatique et d’expulsion qui englobe l’ensemble des pathogènes ESKAPE. Deux des antibiotiques hybrides ont une couverture antibacterienne excellente, et cette étude a mis en évidence des candidats prometteurs pour le développement préclinique. La thérapie avec les antibiotiques aminoglycosidiques est toujours associée à une probabilité de complications néphrotoxiques. Le potentiel de toxicité de chaque aminoglycoside peut être largement corrélé avec le nombre de groupements amino et de désoxygénations. Une hypothèse de longue date dans le domaine indique que les interactions principales sont effectuées par des sels des groupements ammonium, donc l’ajustement des paramètres de pKa pourrait provoquer une dissociation plus rapide avec leurs cibles, une clairance plus efficace et globalement des analogues moins néphrotoxiques. Le Chapitre 5 de cette dissertation présente la conception et la synthèse asymétrique de chaînes N1 HABA β substitutées par mono- et bis-fluoration. Des chaînes qui possèdent des γ-N pKa dans l’intervalle entre 10 et 7.5 ont été appliquées sur une neomycine tétra-désoxygénée pour produire des antibiotiques avancés. Malgré la réduction considérable du γ N pKa, le large spectre bactéricide n’a pas été significativement affecté pour les analogues fluorés isosteriques. De plus, des études structure-toxicité évaluées avec une analyse d’apoptose propriétaire d’Achaogen ont démontré que la nouvelle chaîne β,β difluoro-N1-HABA est moins nocive sur un modèle de cellules de rein humain HK2 et elle est prometteuse pour le développement d’antibiotiques du type neomycine avec des propriétés thérapeutiques améliorées. Le chapitre final de cette dissertation présente la proposition et validation d’une synthèse biomimétique par assemblage spontané du aminoglycoside 66-40C, un dimère C2 symétrique bis-imine macrocyclique à 16 membres. La structure proposée du macrocycle a été affinée par spectroscopie nucléaire à un système trans,trans-bis-azadiène anti-parallèle. Des calculs indiquent que l’effet anomérique de la liaison α glycosidique entre les anneaux A et B fournit la pré-organisation pour le monomère 6’ aldéhydo sisomicine et favorise le produit macrocyclique observé. L’assemblage spontané dans l’eau a été étudié par la dimérisation de trois divers analogues et par des expériences d’entre croisement qui ont démontré la généralité et la stabilité du motif macrocyclique de l'aminoglycoside 66-40C.

Funktionelle Analyse der onkologisch relevanten Protease Threonin-Aspartase 1

Krebs stellt eine der häufigsten Todesursachen in Europa dar. Grundlage für eine langfristige Verbesserung des Behandlungserfolgs ist ein molekulares Verständnis der Mechanismen, welche zur Krankheitsentstehung beitragen. In diesem Zusammenhang spielen Proteasen nicht nur eine wichtige Rolle, sondern stellen auch bei vielerlei Erkrankungen bereits anerkannte Zielstrukturen derzeitiger Behandlungsstrategien dar. Die Protease Threonin Aspartase 1 (Taspase1) spielt eine entscheidende Rolle bei der Aktivierung von Mixed Lineage Leukemia (MLL)-Fusionsproteinen und somit bei der Entstehung aggressiver Leukämien. Aktuelle Arbeiten unterstreichen zudem die onkologische Relevanz von Taspase1 auch für solide Tumore. Die Kenntnisse über die molekularen Mechanismen und Signalnetzwerke, welche für die (patho)biologischen Funktionen von Taspase1 verantwortlich sind, stellen sich allerdings noch immer als bruchstückhaft dar. Um diese bestehenden Wissenslücken zu schließen, sollten im Rahmen der Arbeit neue Strategien zur Inhibition von Taspase1 erarbeitet und bewertet werden. Zusätzlich sollten neue Einsichten in evolutionären Funktionsmechanismen sowie eine weitergehende Feinregulation von Taspase1 erlangt werden. Zum einen erlaubte die Etablierung und Anwendung eines zellbasierten Taspase1-Testsystem, chemische Verbindungen auf deren inhibitorische Aktivität zu testen. Überraschenderweise belegten solch zelluläre Analysen in Kombination mit in silico-Modellierungen eindeutig, dass ein in der Literatur postulierter Inhibitor in lebenden Tumorzellen keine spezifische Wirksamkeit gegenüber Taspase1 zeigte. Als mögliche Alternative wurden darüber hinaus Ansätze zur genetischen Inhibition evaluiert. Obwohl publizierte Studien Taspase1 als ααββ-Heterodimer beschreiben, konnte durch Überexpression katalytisch inaktiver Mutanten kein trans-dominant negativer Effekt und damit auch keine Inhibition des wildtypischen Enzyms beobachtet werden. Weiterführende zellbiologische und biochemische Analysen belegten erstmalig, dass Taspase1 in lebenden Zellen in der Tat hauptsächlich als Monomer und nicht als Dimer vorliegt. Die Identifizierung evolutionär konservierter bzw. divergenter Funktionsmechanismen lieferte bereits in der Vergangenheit wichtige Hinweise zur Inhibition verschiedenster krebsrelevanter Proteine. Da in Drosophila melanogaster die Existenz und funktionelle Konservierung eines Taspase1-Homologs postuliert wurde, wurde in einem weiteren Teil der vorliegenden Arbeit die evolutionäre Entwicklung der Drosophila Taspase1 (dTaspase1) untersucht. Obwohl Taspase1 als eine evolutionär stark konservierte Protease gilt, konnten wichtige Unterschiede zwischen beiden Orthologen festgestellt werden. Neben einem konservierten autokatalytischen Aktivierungsmechanismus besitzt dTaspase1 verglichen mit dem humanen Enzym eine flexiblere Substraterkennungs-sequenz, was zu einer Vergrößerung des Drosophila-spezifischen Degradoms führt. Diese Ergebnisse zeigen des Weiteren, dass zur Definition und Vorhersage des Degradoms nicht nur proteomische sondern auch zellbiologische und bioinformatische Untersuchungen geeignet und notwendig sind. Interessanterweise ist die differentielle Regulation der dTaspase1-Aktivität zudem auf eine veränderte intrazelluläre Lokalisation zurückzuführen. Das Fehlen von in Vertebraten hochkonservierten aktiven Kernimport- und nukleolären Lokalisationssignalen erklärt, weshalb dTaspase1 weniger effizient nukleäre Substrate prozessiert. Somit scheint die für die humane Taspase1 beschriebene Regulation von Lokalisation und Aktivität über eine Importin-α/NPM1-Achse erst im Laufe der Entwicklung der Vertebraten entstanden zu sein. Es konnte also ein bislang unbekanntes evolutionäres Prinzip identifiziert werden, über welches eine Protease einen Transport- bzw. Lokalisations-basierten Mechanismus zur Feinregulation ihrer Aktivität „von der Fliege zum Menschen“ nutzt. Eine weitere Möglichkeit zur dynamischen Funktionsmodulation bieten post-translationale Modifikationen (PTMs) der Proteinsequenz, zu welcher Phosphorylierung und Acetylierung zählen. Interessanterweise konnte für die humane Taspase1 über den Einsatz unabhängiger Methoden einschließlich massenspektrometrischer Analysen eine Acetylierung durch verschiedene Histon-Acetyltransferasen (HATs) nachgewiesen werden. Diese Modifikation erfolgt reversibel, wobei vor allem die Histon-Deacetylase HDAC1 durch Interaktion mit Taspase1 die Deacetylierung der Protease katalysiert. Während Taspase1 in ihrer aktiven Konformation acetyliert vorliegt, kommt es nach Deacetylierung zu einer Reduktion ihrer enzymatischen Aktivität. Somit scheint die Modulation der Taspase1-Aktivität nicht allein über intra-proteolytische Autoaktivierung, Transport- und Interaktionsmechanismen, sondern zudem durch post-translationale Modifikationen gesteuert zu werden. Zusammenfassend konnten im Rahmen dieser Arbeit entscheidende neue Einblicke in die (patho)biologische Funktion und Feinregulation der Taspase1 gewonnen werden. Diese Ergebnisse stellen nicht nur einen wichtigen Schritt in Richtung eines verbesserten Verständnis der „Taspase1-Biologie“, sondern auch zur erfolgreichen Inhibition und Bewertung der krebsrelevanten Funktion dieser Protease dar.

Breast cancer risk and heterocyclic amines and effect modification by NAT gene polymorphisms

Breast cancer is the most common cancer in women in the United States and is a leading cause of cancer-related deaths (1). Recently, dietary heterocyclic amines (HCAs) have been proposed to be a risk factor for breast cancer (2). This study uses the data collected for a case-control study conducted at the M.D. Anderson Cancer Center to assess the association between breast cancer risk and HCAs {2-amino-1-methyl-6-phenylimidazole [4,5-b] pyridine (PhIP), 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo [4,5-f] quinoxaline (DiMeIQx) and mutagenicity of HCAs} and to examine if this association is modified by genetic polymorphisms of N-acetyl transferases (NAT1/NAT2). The NAT1/2 genotype was determined using Taqman technology. HCAs were estimated by using a meat preparation questionnaire on meat type, cooking method, and doneness, combined with a quantitative HCA database. Three hundred and fifty patients with breast cancer attending the Diagnostic Radiology Clinic at M. D. Anderson Cancer Center and fulfilling the eligibility criteria were compared to three hundred and fifty patients attending the same clinic for benign breast lesions to answer these questions. Logistic regression models were used to control for known risk factors and showed no statistically significant association between breast cancer versus benign breast cancer lesions and dietary intake of heterocyclic amines. There was no clear difference in their effect after subgroup analyses in different acetylator strata of NAT1/2 and no statistical interactions were found between NAT1/2 genotypes and HCAs, suggesting no effect modification by NAT1/2 acetylator status. These results suggest the need for further research to analyze if these null associations were because of the benign breast lesions sharing the risk factors with breast cancer or any other factors which haven't been explored yet.^

Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyl-transferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNAT1 mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes.

Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus

Glycolipid glycosyltransferases catalyze the stepwise transfer of monosaccharides from sugar nucleotides to proper glycolipid acceptors. They are Golgi resident proteins that colocalize functionally in the organelle, but their intimate relationships are not known. Here, we show that the sequentially acting UDP-GalNAc:lactosylceramide/GM3/GD3 β-1,4-N-acetyl-galactosaminyltransferase and the UDP-Gal:GA2/GM2/GD2 β-1,3-galactosyltransferase associate physically in the distal Golgi. Immunoprecipitation of the respective epitope-tagged versions expressed in transfected CHO-K1 cells resulted in their mutual coimmunoprecipitation. The immunocomplexes efficiently catalyze the two transfer steps leading to the synthesis of GM1 from exogenous GM3 in the presence of UDP-GalNAc and UDP-Gal. The N-terminal domains (cytosolic tail, transmembrane domain, and few amino acids of the stem region) of both enzymes are involved in the interaction because (i) they reproduce the coimmunoprecipitation behavior of the full-length enzymes, (ii) they compete with the full-length counterpart in both coimmunoprecipitation and GM1 synthesis experiments, and (iii) fused to the cyan and yellow fluorescent proteins, they localize these proteins to the Golgi membranes in an association close enough as to allow fluorescence resonance energy transfer between them. We suggest that these associations may improve the efficiency of glycolipid synthesis by channeling the intermediates from the position of product to the position of acceptor along the transfer steps.

Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology

Although cytosolic glutathione S-transferase (GST) enzymes occupy a key position in biological detoxification processes, two of the most relevant human isoenzymes, GSTT1-1 and GSTM1-1, are genetically deleted (non-functional alleles GSTT1*0 and GSTM1*0) in a high percentage of the human population, with major ethnic differences. The structures of the GSTT and GSTM gene areas explain the underlying genetic processes. GSTT1-1 is highly conserved during evolution and plays a major role in phase-II biotransformation of a number of drugs and industrial chemicals, e.g. cytostatic drugs, hydrocarbons and halogenated hydrocarbons. GSTM1-1 is particularly relevant in the deactivation of carcinogenic intermediates of polycyclic aromatic hydrocarbons. Several lines of evidence suggest that hGSTT1-1 and/or hGSTM1-1 play a role in the deactivation of reactive oxygen species that are likely to be involved in cellular processes of inflammation, ageing and degenerative diseases. There is cumulating evidence that combinations of the GSTM1*0 state with other genetic traits affecting the metabolism of carcinogens (CYP1A1, GSTP1) may predispose the aero-digestive tract and lung, especially in smokers, to a higher risk of cancer. The GSTM1*0 status appears also associated with a modest increase in the risk of bladder cancer, consistent with a GSTM1 interaction with carcinogenic tobacco smoke constituents. Both human GST deletions, although largely counterbalanced by overlapping substrate affinities within the GST superfamily, have consequences when the organism comes into contact with distinct man-made chemicals. This appears relevant in industrial toxicology and in drug metabolism.

Conjugation of carcinogens by 6 class glutathione S-transferases : mechanisms and relevance to variations in human risk

Conjugation of chemicals with glutathione (GSH) can lead to decreased or increased toxicity. A genetic deficiency in the GSH S-transferase μ class gene M1 has been hypothesized to lead to greater risk of lung cancer in smokers. Recently a gene deletion polymorphism involving the human θ enzyme T1 has been described; the enzyme is present in erythrocytes and can be readily assayed. A rat θ class enzyme, 5-5, has structural and catalytic similarity and the protein was expressed in the Salmonella typhimurium tester strain TA1535. Expression of the cDNA vector increased the mutagenicity of ethylene dibromide and several methylene dihalides. Mutations resulting from the known GSH S-transferase substrate 1,2-epoxy-3-(4′nitrophenoxy)propane were decreased in the presence of the transferase. Expression of transferase 5-5 increased mutations when 1,2,3,4-diepoxybutane (butadiene diepoxide), 4-bromo-1,2-epoxybutane, or 1,3-dichloracetone were added. The latter compound is a model for the putative 1,2-dibromo-3-chloropropane oxidation product 1-bromo-3-chloroacetone. These genotoxicity and genotyping assays may be of use in further studies of the roles of GSH S-transferase θ enzymes in bioactivation and detoxication and any changes in risk due to polymorphism.

Influence of polymorphisms of the human glutathione transferases and cytochrome P450 2E1 enzyme on the metabolism and toxicity of ethylene oxide and acrylonitrile

A cohort of 59 persons with industrial handling of low levels of acrylonitrile is being studied as part of a medical surveillance programme. Previously, an extended haemoglobin adduct monitoring (N-(cyanoethyl)valine and N-(hydroxyethyl)-valine) was performed regarding the glutathione transferases hGSTM1 and hGSTT1 polymorphisms but no influence of hGSTM1 or hGSTT1 polymorphisms on specific adduct levels was found. A compilation of case reports of human accidental poisonings had pointed to significant individual differences in human acrylonitrile metabolism and toxicity. Therefore, a re-evaluation of the industrial cohort included known polymorphisms of the glutathione transferases hGSTM3 and hGSTP1 as well as of the cytochrome P450 CYP2E1. A detailed statistical analysis revealed that exposed carriers of the allelic variants of hGSTP1, hGSTP1*B/hGSTP1*C, characterized by a single nucleotide polymorphism at nucleotide 313 which results in a change from Ile to Val at codon 104, had higher levels of the acrylonitrile-specific haemoglobin adduct N-(cyanoethyl)valine compared to the carriers of the codon 113 alleles hGSTP1*A and hGSTP1*D. The single nucleotide polymorphism at codon 113 of hGSTP1 (hGSTP1*A/hGSTP1*B versus hGSTP1*C/hGSTP1*D) did not show an effect, and also no influence was seen on specific haemoglobin adduct levels of the polymorphisms of hGSTM3 or CYP2E1. The data, therefore, point to a possible influence of a human enzyme polymorphism of the GSTP1 gene at codon 104 on the detoxication of acrylonitrile which calls for experimental toxicological investigation. The study also confirmed the impact of GSTT1 polymorphism on background N-(hydroxyethyl)-valine adduct levels in haemoglobin which are caused by endogenous ethylene oxide.

Haemoglobin adducts of acrylonitrile and ethylene oxide in acrylonitrile workers, dependent on polymorphisms of the glutathione transferases GSTT1 and GSTM1

Fifty-nine persons with industrial handling of low levels of acrylonitrile (AN) were studied. As part of a medical surveillance programme an extended haemoglobin adduct monitoring [N-(cyanoethyl)valine, CEV; N- (methyl)valine, MV; N-(hydroxyethyl)valine, HEV] was performed. Moreover, the genetic states of the polymorphic glutathione transferases GSTM1 and GSTT1 were assayed by polymerase chain reaction (PCR). Repetitive analyses of CEV and MV in subsequent years resulted in comparable values (means, 59.8 and 70.3 μg CEV/1 blood; 6.7 and 6.7 μg MV/1 blood). Hence, the industrial AN exposures were well below current official standards. Monitoring the haemoglobin adduct CEV appears as a suitable means of biomonitoring and medical surveillance under such exposure conditions. There was also no apparent correlation between the CEV and HEV or CEV and MV adduct levels. The MV and HEV values observed represented background levels, which apparently are not related to any occupational chemical exposure. There was no consistent effect of the genetic GSTM1 or GSTT1 state on CEV adduct levels induced by acrylonitrile exposure. Therefore, neither GSTM1 nor GSTT1 appears as a major AN metabolizing isoenzyme in humans. The low and physiological background levels of MV were also not influenced by the genetic GSTM1 state, but the MV adduct levels tended to be higher in GSTT1- individuals compared to GSTT1 + persons. With respect to the background levels of HEV adducts observed, there was no major influence of the GSTM1 state, but GST- individuals displayed adduct levels that were about 1/3 higher than those of GSTT1+ individuals. The coincidence with known differences in rates of background sister chromatid exchange between GSTT1- and GSTT1 + persons suggests that the lower ethylene oxide (EO) detoxification rate in GSTT1- persons, indicated by elevated blood protein hydroxyethyl adduct levels, leads to an increased genotoxic effect of the physiological EO background.

5,7-di-N-acetyl-acinetaminic acid: A novel non-2-ulosonic acid found in the capsule of an Acinetobacter baumannii isolate

An Acinetobacter baumannii global clone 1 (GC1) isolate was found to carry a novel capsule biosynthesis gene cluster, designated KL12. KL12 contains genes predicted to be involved in the synthesis of simple sugars, as well as ones for N-acetyl-l-fucosamine (l-FucpNAc) and N-acetyl-d-fucosamine (d-FucpNAc). It also contains a module of 10 genes, 6 of which are required for 5,7-di-N-acetyl-legionaminic acid synthesis. Analysis of the composition of the capsule revealed the presence of N-acetyl-d-galactosamine, l-FucpNAc and d-FucpNAc, confirming the role of fnlABC and fnr/gdr genes in the synthesis of l-FucpNAc and d-FucpNAc, respectively. A non-2-ulosonic acid, shown to be 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-altro-non-2-ulosonic acid, was also detected. This sugar has not previously been recovered from biological source, and was designated 5,7-di-N-acetyl-acinetaminic acid (Aci5Ac7Ac). Proteins encoded by novel genes, named aciABCD, were predicted to be involved in the conversion of 5,7-di-N-acetyl-legionaminic acid to Aci5Ac7Ac. A pathway for 5,7-di-N-acetyl-8-epilegionaminic acid biosynthesis was also proposed. In available A. baumannii genomes, genes for the synthesis of 5,7-di-N-acetyl-acinetaminic acid were only detected in two closely related capsule gene clusters, KL12 and KL13, which differ only in the wzy gene. KL12 and KL13 are carried by isolates belonging to clinically important clonal groups, GC1, GC2 and ST25. Genes for the synthesis of N-acyl derivatives of legionaminic acid were also found in 10 further A. baumannii capsule gene clusters, and three carried additional genes for production of 5,7-di-N-acetyl-8-epilegionaminic acid.

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H2O2, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl(+) cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl(+) CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl(+) cells. NAC enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

Structure of 5'-O-acetyl-2',3'-O-isopropylideneuridine, C14H18N2O7

M r = 326.3, monoclinic, P21, a --= 6.510 (2), b=8.432 (2), c= 15.114 (2),a, /~= 101.42 (3) ° , Z = 2, V= 813.15 A 3, D x = 1-33 Mg m -3, F(000) = 172, 2(Cu Ka) = 1.5418/~,, g(Cu Ka) = 0.906 mm -~, final R = 6.4% for 1924 observed counter reflections. The conformation about the glycosidic bond is syn [torsion angle C(6)-N(1)-C(1')-O(4')=-103.9(3)°]. The sugar pucker is C(2')-exo,C(3')-endo (3Tz). The conformation about the C(4')-C(5') bond is gauche-trans. An uncommon intermolecular hydrogen bond involving the ribose-ring oxygen O(1') and the base-nitrogen N(3) stabilizes the crystal structure.

Pasteurella multocida Expresses Two Lipopolysaccharide Glycoforms Simultaneously, but Only a Single Form Is Required for Virulence: Identification of Two Acceptor-Specific Heptosyl I Transferases

Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-D-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo1 residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures.

4-[2-(1-Acetyl-2-oxopropylidene)-hydrazino]-N-(pyrimidin-2-yl)benzenesulfonamide

In the title compound, C15H15N5O4S, the dihedral angle between the pyrimidine and benzene rings is 84.56 (2)degrees. Intramolecular hydrazine-carbonyl N-H center dot center dot center dot O and intermolecular sulfonamide-pyridimine N-H center dot center dot center dot N hydrogen bonds stabilize the molecular and crystal structures, respectively.