Vascular adaptive responses to physical exercise and to stress are affected differently by nandrolone administration

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Stress amplifies lung tissue mechanics, inflammation and oxidative stress induced by chronic inflammation

Background: Mechanisms linking behavioral stress and inflammation are poorly understood, mainly in distal lung tissue. Objective: We have investigated whether the forced swim stress (FS) could modulate lung tissue mechanics, iNOS, cytokines, oxidative stress activation, eosinophilic recruitment, and remodeling in guinea pigs (GP) with chronic pulmonary inflammation. Methods: The GP were exposed to ovalbumin or saline aerosols (2x/wk/4wks, OVA, and SAL). Twenty-four hours after the 4th inhalation, the GP were submitted to the FS protocol (5x/wk/2wks, SAL-S, and OVA-S). Seventy-two hours after the 7th inhalation, lung strips were cut and tissue resistance (Rt) and elastance (Et) were obtained (at baseline and after OVA and Ach challenge). Strips were submitted to histopathological evaluation. Results: The adrenals' weight, the serum cortisol, and the catecholamines were measured. There was an increase in IL-2, IL-5, IL-13, IFN-gamma, iNOS, 8-iso-PGF2 alpha, and in %Rt and %Et after Ach challenge in the SAL-S group compared to the SAL one. The OVA-S group has had an increase in %Rt and %Et after the OVA challenge, in %Et after the Ach and in IL-4, 8-iso-PGF2 alpha, and actin compared to the OVA. Adrenal weight and cortisol serum were increased in stressed animals compared to nonstressed ones, and the catecholamines were unaltered. Conclusion & clinical relevance: Repeated stress has increased distal lung constriction, which was associated with an increase of actin, IL-4, and 8-iso-PGF2 alpha levels. Stress has also induced an activation of iNOS, cytokines, and oxidative stress pathways.

Transmission of Human Herpesvirus Type 8 Infection Within Families in American Indigenous Populations From the Brazilian Amazon

Background. The intrafamilial dynamics of endemic infection with human herpesvirus type 8 (HHV-8) in Amerindian populations is unknown. Methods. Serum samples were obtained from 517 Amerindians and tested for HHV-8 anti-latent nuclear antigen (anti-LANA) and antilytic antibodies by immunofluorescence assays. Logistic regression and mixed logistic models were used to estimate the odds of being HHV-8 seropositive among intrafamilial pairs. Results. HHV-8 seroprevalence by either assay was 75.4% (95% confidence interval [CI]: 71.5%-79.1%), and it was age-dependent (P-trend<.001). Familial dependence in HHV-8 seroprevalence by either assay was found between mother-offspring (odds ratio [OR], 5.44; 95% CI: 1.62-18.28) and siblings aged >= 10 years (OR 4.42, 95% CI: 1.70-11.45) or siblings in close age range (<5 years difference) (OR 3.37, 95% CI: 1.21-9.40), or in families with large (>4) number of siblings (OR, 3.20, 95% CI: 1.33-7.67). In separate analyses by serological assay, there was strong dependence in mother-offspring (OR 8.94, 95% CI: 2.94-27.23) and sibling pairs aged >= 10 years (OR, 11.91, 95% CI: 2.23-63.64) measured by LANA but not lytic antibodies. Conclusions. This pattern of familial dependence suggests that, in this endemic population, HHV-8 transmission mainly occurs from mother to offspring and between close siblings during early childhood, probably via saliva. The mother to offspring dependence was derived chiefly from anti-LANA antibodies.

Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1��� (HIF-1���) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5���aHIF-1���) antisense to the human HIF-1��� mRNA and a known antisense RNA at the 3��� end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1��� and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the ���Topo-Seq��� approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5��� and 3��� ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3��� ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5��� ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

Alkylantien induzierte Zytotoxizit��t, DNA-schadensinduzierte Reparaturkapazit��t und Apoptoseinduktion von Immunzellen

Monozyten und Monozyten-abgeleitete Dendritische Zellen (DCs) spielen eine bedeutende Rolle im Immunsystem. Da DCs bei der Tumorabwehr mitwirken, ist es wichtig, dass Monozyten als auch DCs sich gegen��ber zytotoxischen Agenzien aus der Chemotherapie wehren k��nnen. Chemotherapeutika reagieren mit der DNA, jedoch die DNA-Reparaturkapazit��t von Monozyten und DCs wurde noch nicht untersucht. Dazu wurde die Sensitivit��t in Monozyten und DCs gegen��ber verschiedene genotoxische Agenzien untersucht. Dabei wurde herausgefunden, dass Monozyten sensitiv auf methylierende Agenzien (MNNG, MMS und Temozolomid) reagieren und ein verst��rktes Zellsterben und Apoptoseinduktion zeigen. Im Vergleich zu weiteren Zytostatika wie Fotemustin, Mafosfamid und Cisplatin reagierten Monozyten und DCs gleich sensitiv. Diese Ergebnisse weisen auf einen Defekt in der Reparatur von DNA-Methylierungssch��den in Monozyten hin. Da die Expression des Reparaturproteins O6-Methylguanin-DNA Methyltransferase (MGMT) in Monozyten h��her war als in DCs und deren Inhibierung durch O6-Benzylguanin keinen Effekt auf die Sensitivit��t von Monozyten hatte, wurde der Reparaturweg der Basenexzisionsreparatur untersucht. Im Vergleich zu DCs waren die Monozyten unf��hig die BER durchzuf��hren, welche durch Einzelzellgelelektrophorese gemessen wurde. Expressionsuntersuchungen ergaben, dass in Monozyten XRCC1 und Ligase III�� fehlen im Vergleich zu DCs, Makrophagen, h��matopoetische Stammzellen und Lymphozyten, welche diese Proteine exprimieren. Diese Ergebnisse zeigen einen spezifischen DNA-Reparaturdefekt in einer bestimmten Blutzellpopulation. Durch den BER Defekt in Monozyten kann es durch methylierende Tumorwirkstoffe w��hrend einer Chemotherapie zur Depletion und zu einer abgeschw��chten Immunantwort kommen.

Relevanz der Methioninoxidation aus evolution��rer Sicht und im akuten oxidativen Stressmodell der Alzheimerschen Krankheit

Im Laufe der Evolution m��ssen Sauerstoff-metabolisierende Organismen eine Reihe von Anpassungen entwickelt haben, um in der zytotoxischen oxidativen Umgebung der sauerstoff-haltigen Erdatmosph��re ��berleben zu k��nnen. Die im Rahmen dieser Arbeit durchgef��hrten vergleichenden Analysen mitochondrial kodierter und kern-kodierter Proteome mehrerer hundert Spezies haben ergeben, dass die Evolution eines alternativen genetischen Codes in Mitochondrien eine moderne Adaptation in diesem Sinne war. Viele aerobe Tiere und Pilze dekodieren in Abweichung vom genetischen Standard-Code das Codon AUA als Methionin. In der vorliegenden Arbeit wird gezeigt, dass diese Spezies dadurch eine massive Akkumulation der sehr leicht oxidierbaren Aminos��ure Methionin in ihren Atmungskettenkomplexen erreichen, die generell ein bevorzugtes Ziel reaktiver Sauerstoffspezies sind. Der gewonnene Befund l��sst sich widerspruchsfrei nur unter Annahme einer antioxidativen Wirkung dieser Aminos��ure erkl��ren, wie sie erstmals 1996 von R. Levine anhand von Oxidationsmessungen in Modellproteinen postuliert worden war. In der vorliegenden Arbeit wird diese Hypothese nun direkt mittels neuartiger Modellsubstanzen in lebenden Zellen best��tigt. Die durchgef��hrten bioinformatischen Analysen und zellbiologischen Experimente belegen, dass kollektive Proteinver��nderungen die Triebkraft f��r die Evolution abweichender genetischer Codes sein k��nnen.rnDie Bedeutung von oxidativem Stress wurde dar��ber hinaus auch im Referenzrahmen einer akuten oxidativen Sch��digung im Einzelorganismus untersucht. Da oxidativer Stress in der Pathogenese altersassoziierter neurodegenerativer Erkrankungen wie der Alzheimerschen Krankheit prominent involviert zu sein scheint, wurden die Auswirkungungen von Umwelt-induziertem oxidativem Stress auf den histopathologischen Verlauf in einem transgenen Modell der Alzheimerschen Krankheit in vivo untersucht. Dabei wurden transgene M��use des Modells APP23 im Rahmen von F��tterungsversuchen einer lebenslangen Defizienz der Antioxidantien Selen oder Vitamin E ausgesetzt. W��hrend die Selenoproteinexpression durch die selendefiziente Di��t gewebespezifisch reduziert wurde, ergaben sich keine Anzeichen eines beschleunigten Auftretens pathologischer Marker wie amyloider Plaques oder Neurodegeneration. Es war vielmehr ein unerwarteter Trend hinsichtlich einer geringeren Plaquebelastung in Vitamin E-defizienten Alzheimerm��usen zu erkennen. Auch wenn diese Daten aufgrund einer geringen Versuchstiergruppengr����e nur mit Vorsicht interpretiert werden d��rfen, so scheint doch ein Mangel an essentiellen antioxidativen N��hrstoffen die Progression in einem anerkannten Alzheimermodell nicht negativ zu beeinflussen.rn

Expression und Funktion der Paraoxonase-2 im Hinblick auf oxidativen Stress im Gef����system

Erh��hte Spiegel von oxidativem Stress bedingen Atherosklerose, eine Krankheit die ��ber 50% aller Todesf��lle in der westlichen Welt ausmacht. Es ist entscheidend Mechanismen zur Abwehr dieser Krankheit zu ergr��nden.rnDa genetische Polymorphismen des k��rpereigenen Enzyms Paraoxonase 2 (PON2) mit kardiovaskul��ren Erkrankungen assoziiert sind, wurden ihre Regulation und potentiell antioxidativen Funktionen in vaskul��ren Zellen analysiert. Mittels verschiedener molekularbiologischer Methoden konnte ich erstmals zeigen, dass PON2 in vaskul��ren Zellen vornehmlich subzellul��r im ER lokalisiert ist. Anhand verschiedener Experimente wurde PON2 als potenter Faktor zur Reduktion von ROS identifiziert. Erh��hte ROS-Spiegel f��hren zur Aktivierung eines als unfolded protein response (UPR) bekannten ER-Stress-Signalwegs. Dieser ist neben Atherosklerose in eine Vielzahl von Erkrankungen involviert und hat kritischen Einfluss auf das ��berleben oder Absterben von Zellen. Durchgef��hrte Promoter-Reporter Studien bewiesen die Induktion der Protein-Expression von PON2 nach Aktivierung des UPR-Signalwegs, was als kompensatorischer Mechanismus der Zelle zur Vermeidung UPR-induzierter Apoptose verstanden werden k��nnte. PON2 wehrt oxidativen Stress und die UPR-induzierte Apoptose ab und ist ein protektiver Faktor vor Atherosklerose.rnIn einem Krebsmodell k��nnte PON2 aber als antiapoptotischer Faktor entscheidend am ��berleben von Tumorzellen beteiligt sein. Gerade diese beiden gegens��tzlichen Aspekte der antiapoptotischen Funktion des Proteins zeigen die Notwendigkeit f��r weitere Untersuchungen zu PON2 auf.rn

Untersuchung der Rolle reaktiver Sauerstoffspezies und DNA-Sch��den bei Hypertonie und Arteriosklerose

Angiotensin II induziert intrazellul��r die Bildung reaktiver Sauerstoffspezies, welche DNA-Sch��den erzeugen k��nnen. Um die Hypothese zu pr��fen, dass durch Angiotensin II induzierte DNA-Sch��den f��r die erh��hte Krebsinzidenz hypertensiver Menschen verantwortlich sind, wurde eine vierw��chige Behandlung von M��usen mit Angiotensin II (0,6 ��g/kg/min) durchgef��hrt. Mit der Alkalischen Elution wurden in Zellen aus verschiedenen Organen der M��use die Menge an DNA-Einzelstrangbr��chen und oxidativen DNA-Modifikationen bestimmt. In der Niere wurde au��erdem mit dem BigBlue�� Mutations-Assay die Entstehung von Mutationen analysiert. In keinem der analysierten Organe konnte eine Erh��hung der DNA-Sch��den oder eine Erh��hung der Mutationsfrequenzen durch die Angiotensin II-Behandlung nachgewiesen werden. Die durchgef��hrten Untersuchungen geben somit keinen Hinweis auf eine DNA-sch��digende und mutagene Wirkung von Angiotensin II.rnBei der Entstehung und dem Krankheitsverlauf von Arteriosklerose spielen reaktive Sauerstoffspezies ebenfalls eine noch nicht genau gekl��rte Rolle. Um zu ermitteln, ob oxidative DNA-Sch��den die Entstehung der Arteriosklerose beg��nstigen, wurde die Endothelfunktion von Wildtyp- und reparaturdefizienten Ogg1-/--M��usen verglichen. Entgegen der Vermutung, dass oxidative DNA-Modifikationen die Endothelfunktion verschlechtern, zeigen die Untersuchungen, dass Ogg1-/--M��use, die h��here Spiegel an oxidativen DNA-Modifikationen in ihrem Genom haben, eine signifikant bessere Endothelfunktion besitzen als Wildtyptiere. Dieser Befund weist auf eine neuartige, von der DNA-Reparatur unabh��ngige Funktion von OGG1 hin.rn

Die Bedeutung von molekularem Sauerstoff f��r die Evolution des genetischen Codes

Die Entstehung und Evolution des genetischen Codes, der die Nukleotidsequenz der mRNA in die Aminos��uresequenz der Proteine ��bersetzt, z��hlen zu den gr����ten R��tseln der Biologie. Die ersten Organismen, die vor etwa 3,8 Milliarden Jahren auf der Erde auftraten, nutzten einen urspr��nglichen genetischen Code, der vermutlich ausschlie��lich abiotisch verf��gbare Aminos��uren terrestrischer oder extraterrestrischer Herkunft umfasste. Neue Aminos��uren wurden sukzessive biosynthetisiert und selektiv in den Code aufgenommen, welcher in der modernen Form aus bis zu 22 Aminos��uren besteht. Die Ursachen f��r die Selektion und die Chronologie ihrer Aufnahme sind bis heute unbekannt und sollten im Rahmen der vorliegenden Arbeit erforscht werden. Auf Grundlage quanten-chemischer Berechnungen konnte in dieser Arbeit zun��chst ein Zusammenhang zwischen der HOMO-LUMO-Energiedifferenz (H-L-Distanz), die ein inverses quanten-chemisches Korrelat f��r allgemeine chemische Reaktivit��t darstellt, und der chronologischen Aufnahme der Aminos��uren in den genetischen Code aufgezeigt werden. Demnach sind urspr��ngliche Aminos��uren durch gro��e H-L-Distanzen und neue Aminos��uren durch kleine H-L-Distanzen gekennzeichnet. Bei einer Analyse des Metabolismus von Tyrosin und Tryptophan, bei denen es sich um die beiden j��ngsten Standard-Aminos��uren handelt, wurde ihre Bedeutung als Vorl��ufer von Strukturen ersichtlich, die sich durch eine hohe Redox-Aktivit��t auszeichnen und deren Synthese gleichzeitig molekularen Sauerstoff erfordert. Aus diesem Grund wurden die Redox-Aktivit��ten der 20 Standard-Aminos��uren gegen��ber Peroxylradikalen und weiteren Radikalen getestet. Die Untersuchungen ergaben eine Korrelation zwischen evolution��rem Auftreten und chemischer Reaktivit��t der jeweiligen Aminos��ure, die sich insbesondere in der effizienten Reaktion zwischen Tryptophan bzw. Tyrosin und Peroxylradikalen widerspiegelte. Dies indizierte eine potentielle Bedeutung reaktiver Sauerstoffspezies (ROS) bei der Konstituierung des genetischen Codes. Signifikante Mengen an ROS wurden erst zu Beginn der Oxygenierung der Geobiosph��re, die als Great Oxidation Event (GOE) bezeichnet wird und vor circa 2,3 Milliarden Jahren begann, gebildet und m��ssen zur oxidativen Sch��digung vulnerabler, zellul��rer Strukturen gef��hrt haben. Aus diesem Grund wurde das antioxidative Potential von Aminos��uren beim Prozess der Lipidperoxidation untersucht. Es konnte gezeigt werden, dass lipophile Derivate von Tryptophan und Tyrosin bef��higt sind, die Peroxidation von Rattenhirnmembranen zu verhindern und humane Fibroblasten vor oxidativem Zelltod zu sch��tzen. Daraus gr��ndete sich das in dieser Arbeit aufgestellte Postulat eines Selektionsvorteils primordialer Organismen w��hrend des GOEs, die Tryptophan und Tyrosin als redox-aktive Aminos��uren in Membranproteine einbauen konnten und somit vor Oxidationsprozessen gesch��tzt waren. Demzufolge wurde die biochemische Reaktivit��t als Selektionsparameter sowie oxidativer Stress als pr��gender Faktor der Evolution des genetischen Codes identifiziert.

INDUCTION OF DNA STRAND BREAKS AND CROSS-LINKS BY THE NEW ANTICANCER AGENT 3,6-DIAZIRIDINYL-2,5-BIS(CARBOETHOXYAMINO) -1,4-BENZOQUINONE

The DNA breakage effect of the anticancer agent 3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone (AZQ, NSC-182986) on bacteriophage PM2 DNA was investigated using agarose gel electrophoresis. AZQ caused both single-stranded and double-stranded breaks after reduction with NaBH(,4), but it was not active in the native state. At 120 (mu)M, it degraded 50% of the closed circular form I DNA into 40% form II DNA (single-stranded break) and 10% form III DNA (double-stranded break). It produced a dose-response breakage between 1 (mu)M and 320 (mu)M. The DNA breakage exhibited a marked pH dependency. At 320 (mu)M, AZQ degraded 80% and 60% of form I DNA at pH 4 and 10 respectively, but none between pH 6 to 8. The DNA breakage at physiologic pH was greatly enhanced when 10 (mu)M cupric sulfate was included in the incubation mixture. The DNA strand scission was inhibited by catalase, glutathione, KI, histidine, Tiron, and DABCO. These results suggest that the DNA breakage may be caused by active oxygen metabolites including hydroxyl free radical. The bifunctional cross-linking activity of reduced AZQ on isolated calf thymus DNA was investigated by ethidium fluorescence assay. The cross-linking activity exhibited a similar pH dependency; highest in acidic and alkaline pH, inactive under neutral conditions. Using the alkaline elution method, we found that AZQ induced DNA single-stranded breaks in Chinese hamster ovary cells treated with 50 (mu)M of AZQ for 2 hr. The single-stranded break frequencies in rad equivalents were 17 with 50 (mu)M and 140 with 100 (mu)M of AZQ. In comparison, DNA cross-links appeared in cells treated with only 1 to 25 (mu)M of AZQ for 2 hr. The cross-linking frequencies in rad equivalents were 39 and 90 for 1 and 5 (mu)M of AZQ, respectively. Both DNA-DNA and DNa-protein cross-links were induced by AZQ in CHO cells as revealed by the proteinas K digestion assay. DNA cross-links increased within the first 4 hr of incubation in drug-free medium and slightly decreased by 12 hr, and most of the cross-links disappeared after cells were allowed to recovered for 24 hr.^ By electrochemical analysis, we found that AZQ was more readily reduced at acidic pH. However, incubation of AZQ with NaBH(,4) at pH 7.8 or 10, but not at 4, produced superoxide anion. The opening of the aziridinyl rings of AZQ at pH 4 was faster in the presence of NaBH(,4) than in its absence; no ring-opening was detected at pH 7.8 regardless of the inclusion of NaBH(,4). . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase

3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glycosylase not only for the cytotoxic 3MeA DNA lesion, but also for the mutagenic 1,N6-ethenoadenine (��A) and hypoxanthine lesions. Aag appears to be the only 3MeA and hypoxanthine DNA glycosylase in liver, testes, kidney, and lung, and the only ��A DNA glycosylase in liver, testes, and kidney; another ��A DNA glycosylase may be expressed in lung. Although alkyladenine DNA glycosylase has the capacity to remove 8-oxoguanine DNA lesions, it does not appear to be the major glycosylase for 8-oxoguanine repair. Fibroblasts derived from Aag ���/��� mice are alkylation sensitive, indicating that Aag ���/��� mice may be similarly sensitive.

Mechanism of stimulation of the DNA glycosylase activity of hOGG1 by the major human AP endonuclease: bypass of the AP lyase activity step

The generation of reactive oxygen species in the cell��provokes, among other lesions, the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic. To minimise the mutagenic potential of this oxidised purine, human cells have a specific 8-oxoG DNA glycosylase/AP lyase (hOGG1) that initiates the base excision repair (BER) of 8-oxoG. We show here that in vitro this first enzyme of the BER pathway is relatively inefficient because of a high affinity for the product of the reaction it catalyses (half-life of the complex is >2 h), leading to a lack of hOGG1 turnover. However, the glycosylase activity of hOGG1 is stimulated by the major human AP endonuclease, HAP1 (APE1), the enzyme that performs the subsequent step in BER, as well as by a catalytically inactive mutant (HAP1-D210N). In the presence of HAP1, the AP sites generated by the hOGG1 DNA glycosylase can be occupied by the endonuclease, avoiding the re-association of hOGG1. Moreover, the glycosylase has a higher affinity for a non-cleaved AP site than for the cleaved DNA product generated by HAP1. This would shift the equilibrium towards the free glycosylase, making it available to initiate new catalytic cycles. In contrast, HAP1 does not affect the AP lyase activity of hOGG1. This stimulation of only the hOGG1 glycosylase reaction accentuates the uncoupling of its glycosylase and AP lyase activities. These data indicate that, in the presence of HAP1, the BER of 8-oxoG residues can be highly efficient by bypassing the AP lyase activity of hOGG1 and thus excluding a potentially rate limiting step.

Oxidative DNA Damage Modulates Trinucleotide Repeat Instability Via DNA Base Excision Repair

Trinucleotide repeat (TNR) expansion is the cause of more than 40 types of human neurodegenerative diseases such as Huntington���s disease. Recent studies have linked TNR expansion with oxidative DNA damage and base excision repair (BER). In this research, we provided the first evidence that oxidative DNA damage can induce CAG repeat deletion/contraction via BER. We found that BER of an oxidized DNA base lesion, 8-oxoguanine in a CAG repeat tract, resulted in the formation of a CTG hairpin at the template strand. DNA polymerase �� (pol b) then skipped over the hairpin creating a 5���-flap that was cleaved by flap endonuclease 1 (FEN1) leading to CAG repeat deletion. To further investigate whether BER may help to shorten an expanded TNR tract, we examined BER in a CAG repeat hairpin loop. We found that 8-oxoguanine DNA glycosylase removed the oxidized base located in the loop region of the hairpin leaving an abasic site. Apurinic/apyrimidinic (AP) endonuclease 1 then incised the 5���-end of the abasic site leaving a nick in the loop. This further converted the hairpin into an intermediate with a 3���-flap and a 5���-flap. As a 5���-3��� endonuclease, FEN1 cleaved the 5���-flap, whereas a 3���-5��� endonuclease, Mus81/Eme1, removed the 3���-flap. The coordination between FEN1 and Mus81/Eme1 ultimately resulted in removal of a CAG repeat hairpin attenuating or preventing TNR expansion. To further explore if pol �� bypass of an oxidized base lesion, 5���,8-cyclodeoxyadenosine, may affect TNR instability, we examined pol �� DNA synthesis in bypassing this base lesion and found that the lesion preferentially induced TNR deletion during BER and Okazaki fragment maturation. The repeat deletion was mediated by the formation of a loop in the template strand induced specifically by the damage. Pol �� then skipped over the loop structure creating a 5���-flap that was efficiently removed by FEN1 leading to repeat deletion. Our study demonstrates that pol ��-mediated BER plays an important role in mediating TNR deletion and removing a TNR hairpin to prevent TNR expansion. Our research provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

Prediction of outcomes in hypertensive patients with suspected coronary disease

Stress echocardiography has been shown to improve the diagnosis of coronary artery disease in the presence of hypertension, but its value in prognostic evaluation is unclear. We sought to determine whether stress echocardiography could be used to predict mortality in 2363 patients with hypertension, who were followed for up to 10 years (mean 4.0+/-1.8) for death and revascularization. Stress echocardiograms were normal in 1483 patients (63%), 16% had resting left ventricular (LV) dysfunction alone, and 21% had ischemia. Abnormalities were confined to one territory in 489 patients (21%) and to multiple territories in 365 patients (15%). Cardiac death was less frequent among the patients able to exercise than among those undergoing dobutamine echocardiography (4% versus 7%, P<0.001). The risk of death in patients with a negative stress echocardiogram was <1% per year. Ischemia identified by stress echocardiography was an independent predictor of mortality in those able to exercise (hazard ratio 2.21, 95% confidence intervals 1.10 to 4.43, P=0.0001) as well as those undergoing dobutamine echo (hazard ratio 2.39, 95% confidence intervals 1.53 to 3.75, P=0.0001); other predictors were age, heart failure, resting LV dysfunction, and the Duke treadmill score. In stepwise models replicating the sequence of clinical evaluation, the results of stress echocardiography added prognostic power to models based on clinical and stress-testing variables. Thus, the results of stress echocardiography are an independent predictor of cardiac death in hypertensive patients with known or suspected coronary artery disease, incremental to clinical risks and exercise results.

New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach

Malaria, caused by Plasmodium falciparum (P. falciparum), ranks as one of the most baleful infectious diseases worldwide. New antimalarial treatments are needed to face existing or emerging drug resistant strains. Protein degradation appears to play a significant role during the asexual intraerythrocytic developmental cycle (IDC) of P. falciparum. Inhibition of the ubiquitin proteasome system (UPS), a major intracellular proteolytic pathway, effectively reduces infection and parasite replication. P. falciparum and erythrocyte UPS coexist during IDC but the nature of their relationship is largely unknown. We used an approach based on Tandem Ubiquitin-Binding Entities (TUBEs) and 1D gel electrophoresis followed by mass spectrometry to identify major components of the TUBEs-associated ubiquitin proteome of both host and parasite during ring, trophozoite and schizont stages. Ring-exported protein (REX1), a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, was found to reach a maximum level of ubiquitylation in trophozoites stage. The Homo sapiens (H. sapiens) TUBEs associated ubiquitin proteome decreased during the infection, whereas the equivalent P. falciparum TUBEs-associated ubiquitin proteome counterpart increased. Major cellular processes such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection.