Clinical Heart fAilure Management Program: Changing the practice by partnering primary care and specialists (CHAMP-HF).

Background: While significant gains were made in the management of heart failure (HF), most patients are still diagnosed when they are acutely ill in hospital, often with advanced disease. Earlier diagnosis in the community could lead to improved outcomes. Whether a partnership and an educational program for primary care providers (PCP) increase HF awareness and management is unknown. Methods: We conducted an observational study between March 2019 and June 2020 during which HF specialists gave monthly HF conferences to PCP. Using a pre-post design, medical charts and administrative databases were reviewed and a questionnaire was completed by participating PCP. Primary and secondary endpoints included: 1) the number of patients diagnosed with HF, 2) implementation of GDMT for patients with HFrEF; 3) PCPs' experience and confidence. Results: Six PCP agreed to participate. Amongst the 11,909 patients of the clinic, 70 (0.59 %) patients met the criteria for HF. This number increased by 28.6 % (n = 90) after intervention. Increased use of GDMT for HFrEF patients at baseline (n = 35) was observed for all class of agents, with doubling of patients on triple therapies, from 8 (22.9 %) to 16 (45.7 %), p = 0.0047. Self-confidence on HF management was low (1, 16.7 %) but increased after the educational intervention of physicians (3, 50 %). Conclusion: An educational and collaborative approach between HF specialists and community PCP increased the number of new HF cases diagnosed, enhanced implementation of GDMT in patients with HFrEF and increase PCPs' confidence in treating HF, despite being conducted during the COVID-19 pandemic.

Rapamycin-loaded Poly(lactic-co-glycolic) acid nanoparticles: Preparation, characterization, and in vitro toxicity study for potential intra-articular injection.

Osteoarthritis (OA) is the most common degenerative joint disease. Rapamycin is a potential candidate for OA treatment by increasing the autophagy process implicated in its physiopathology. To optimize Rapamycin profit and avoid systemic side effects, intra-articular (i.a.) administration appeared helpful. However, Rapamycin's highly hydrophobic nature and low bioavailability made it challenging to develop purpose-made drug delivery systems to overcome these limitations. We developed Rapamycin-loaded nanoparticles (NPs) using poly (lactic-co-glycolic acid) by emulsion/evaporation method. We evaluated these NPs' cytocompatibility towards cartilage (chondrocytes) and synovial membrane cells (synoviocytes) for a potential i.a. administration. The in vitro characterization of Rapamycin-loaded NPs had shown a suitable profile for an i.a. administration. In vitro biocompatibility of NPs was highlighted to 10 µM of Rapamycin for both synoviocytes and chondrocytes, but significant toxicity was observed with higher concentrations. Besides, synoviocytes are more sensitive to Rapamycin-loaded NPs than chondrocytes. Finally, we observed in vitro that an adapted formulated Rapamycin-loaded NPs could be safe at suitable i.a. injection concentrations. The toxic effect of Rapamycin encapsulated in these NPs on both articular cells was dose-dependent. After Rapamycin-loaded NPs i.a. administration, local retention, in situ safety, and systemic release should be evaluated with experimental in vivo models.

Estimating the dispersal of Lepeophtheirus salmonis sea lice within and among Atlantic salmon sites of the Bay of Fundy, New Brunswick.

The objective of this study was to estimate the impact of infestation pressures on the abundance of the parasitic sea louse, Lepeophtheirus salmonis, in the Bay of Fundy, New Brunswick (NB), Canada, using the Fish-iTrends database for the years 2009-2018. Infestation pressures were calculated as time-lagged weighted averages of the abundance of adult female (AF) sea lice within a site (internal infestation pressure: IIP) and among sites (external infestation pressure: EIP). The EIP weights were calculated from seaway distances among sites and a Gaussian kernel density for bandwidths of 5 to 60 km. The EIP with a bandwidth of 10 km had the best fit, as determined with Akaike's information criterion, and historical AF sea lice abundance. This estimated dispersal distance of 10 km was similar to previous studies in Norway, Scotland and in New Brunswick. The infestation pressures estimated from empirical AF sea lice abundance within and among sites significantly increased the abundance of AF sea lice (p < .001). This study concludes that sea lice burdens within Atlantic salmon farms in the Bay of Fundy, NB, are affected by within site management and could be improved by synchronizing treatments between sites.

Challenging development of storable particles for oral delivery of a physiological nitric oxide donor.

Nitric oxide (NO) deficiency is often associated with several acute and chronic diseases. NO donors and especially S-nitrosothiols such as S-nitrosoglutathione (GSNO) have been identified as promising therapeutic agents. Although their permeability through the intestinal barrier have recently be proved, suitable drug delivery systems have to be designed for their oral administration. This is especially challenging due to the physico-chemical features of these drugs: high hydrophilicity and high lability. In this paper, three types of particles were prepared with an Eudragit® polymer: nanoparticles and microparticles obtained with a water-in-oil-in-water emulsion/evaporation process versus microparticles obtained with a solid-in-oil-in-water emulsion/evaporation process. They had a similar encapsulation efficiency (around 30%), and could be freeze-dried then be stored at least one month without modification of their critical attributes (size and GSNO content). However, microparticles had a slightly slower in vitro release of GSNO than nanoparticles, and were able to boost by a factor of two the drug intestinal permeability (Caco-2 model). Altogether, this study brings new data about GSNO intestinal permeability and three ready-to-use formulations suitable for further preclinical studies with oral administration.

Understanding the Thermodynamic Mechanisms Leading to the Binding of Albumin to Lipid Nanocapsules.

Lipid nanocapsules (LNCs) are drug delivery platforms designed for different administration routes including intravenous delivery. Nanocarrier binding with plasma proteins such as albumin is an important factor that influences the pharmacokinetics of the drug and the drug delivery system. The aim of this paper was to characterize LNCs with different surface compositions and hydrophobicities to study their interactions with albumin: binary LNCs [oil-glyceryl trioctanoate (TG) and PEGylated surfactant macrogol 15-hydroxystearate (MHS)] and ternary LNCs (TG, MHS, and Span 80). Span was found to stabilize and decrease the LNC size. The formation of a stable LNC/albumin complex in the ground state was demonstrated. Thermodynamic parameters indicated that complex formation was exothermic and spontaneous, and the interactions involved van der Waals forces and hydrogen bond formation. Ternary LNCs showed higher affinity for albumin than did binary LNCs (affinity constant 10-fold higher). This study is the first report on the thermodynamic mechanisms that lead to the formation of a complex between albumin and organic nanoparticles with different surface architectures.

S-Nitrosothiols as potential therapeutics to induce a mobilizable vascular store of nitric oxide to counteract endothelial dysfunction.

Endothelial dysfunction predisposing to cardiovascular diseases is defined as an imbalance in the production of vasodilating factors, such as nitric oxide (NO), and vasoconstrictive factors. To insure its physiological role, NO, a radical with very short half-life, requires to be stored and transported to its action site. S-nitrosothiols (RSNOs) like S-nitrosoglutathione (GSNO) represent the main form of NO storage within the vasculature. The NO store formed by RSNOs is still bioavailable to trigger vasorelaxation. In this way, RSNOs are an emerging class of NO donors with a potential to restore NO bioavailability within cardiovascular disorders. The aim of this study was to compare S-nitrosothiols ability, formed of peptide (glutathione) like the physiologic GSNO or derived from amino acids (cysteine, valine) like the synthetics S-nitroso-N-acetylcysteine (NACNO) and S-nitroso-N-acetylpenicillamine (SNAP), respectively, to produce a vascular store of NO either in endothelium-intact or endothelium-removed aortae in order to evaluate whether RSNOs can be used as therapeutics to compensate endothelial dysfunction. Sodium nitroprusside (SNP), a marketed drug already in clinics, was used as a non-RSNO NO-donor. Endothelium-intact or endothelium-removed aortae, isolated from normotensive Wistar rats, were exposed to RSNOs or SNP. Then, NO-derived (NOx) species, representing the NO store inside the vascular wall, were quantified using the diaminonaphthalene probe coupled to mercuric ions. The bioavailability of the NO store and its ability to induce vasodilation was tested using N-acetylcysteine, then its ability to counteract vasoconstriction was challenged using phenylephrine (PHE). All the studied RSNOs were able to generate a NO store materialized by a three to five times increase in NOx species inside aortae. NACNO was the most potent RSNO to produce a vascular NO store bioavailable for vasorelaxation and the most efficient to induce vascular hyporeactivity to PHE in endothelium-removed aortae. GSNO and SNAP were equivalent and more efficient than SNP. In endothelium-intact aortae, the NO store was also formed whereas it seemed less available for vasorelaxation and did not influence PHE-induced vasoconstriction. In conclusion, RSNOs - NACNO in a better extent - are able to restore NO bioavailability as a functional NO store within the vessel wall, especially when the endothelium is removed. This was associated with a hyporeactivity to the vasoconstrictive agent phenylephrine. Treatment with RSNOs could present a benefit to restore NO-dependent functions in pathological states associated with injured endothelium.

Intestinal absorption of S-nitrosothiols: Permeability and transport mechanisms.

S-Nitrosothiols, a class of NO donors, demonstrate potential benefits for cardiovascular diseases. Drugs for such chronic diseases require long term administration preferentially through the oral route. However, the absorption of S-nitrosothiols by the intestine, which is the first limiting barrier for their vascular bioavailability, is rarely evaluated. Using an in vitro model of intestinal barrier, based on human cells, the present work aimed at elucidating the mechanisms of intestinal transport (passive or active, paracellular or transcellular pathway) and at predicting the absorption site of three S-nitrosothiols: S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-l-cysteine (NACNO) and S-nitroso-N-acetyl-d-penicillamine (SNAP). These S-nitrosothiols include different skeletons carrying the nitroso group, which confer different physico-chemical characteristics and biological activities (antioxidant and anti-inflammatory). According to the values of apparent permeability coefficient, the three S-nitrosothiols belong to the medium class of permeability. The evaluation of the bidirectional apparent permeability demonstrated a passive diffusion of the three S-nitrosothiols. GSNO and NACNO preferentially cross the intestinal barrier though the transcellular pathway, while SNAP followed both the trans- and paracellular pathways. Finally, the permeability of NACNO was favoured at pH 6.4, which is close to the pH of the jejunal part of the intestine. Through this study, we determined the absorption mechanisms of S-nitrosothiols and postulated that they can be administrated through the oral route.

Glutathione: Antioxidant Properties Dedicated to Nanotechnologies.

Which scientist has never heard of glutathione (GSH)? This well-known low-molecular-weight tripeptide is perhaps the most famous natural antioxidant. However, the interest in GSH should not be restricted to its redox properties. This multidisciplinary review aims to bring out some lesser-known aspects of GSH, for example, as an emerging tool in nanotechnologies to achieve targeted drug delivery. After recalling the biochemistry of GSH, including its metabolism pathways and redox properties, its involvement in cellular redox homeostasis and signaling is described. Analytical methods for the dosage and localization of GSH or glutathiolated proteins are also covered. Finally, the various therapeutic strategies to replenish GSH stocks are discussed, in parallel with its use as an addressing molecule in drug delivery.

One-week in vivo sustained release of a peptide formulated into in situ forming implants.

The LR12 peptide has been reported to reduce the size of infarct and improve both cardiac function and survival in myocardial infarction in murine models, after daily repeated intraperitoneal injections. In order to protect peptide from degrading and to prolong its release, in situ implants based on biocompatible biodegradable polymers were prepared and both in vitro and in vivo releases were evaluated after subcutaneous administration to Wistar rats. A progressive and complete release was obtained in vitro in 3 weeks. In vivo, a 7-day sustained release was demonstrated after administrating the formulation once; bioavailability was improved by protecting the peptide against the degradation identified as a dimerization through disulfide bond formation. As a conclusion, in situ forming formulations are a suitable alternative for the therapeutic use of this peptide.

Design of calcium phosphate ceramics for drug delivery applications in bone diseases: A review of the parameters affecting the loading and release of the therapeutic substance.

Effective treatment of critical-size defects is a key challenge in restorative surgery of bone. The strategy covers the implantation of biocompatible, osteoconductive, bioactive and biodegradable devices which (1) well interact with native tissue, mimic multi-dimensional and hierarchical structure of bone and (2) are able to enhance bone repair, treating post implantation pathologies or bone diseases by local delivery of therapeutic agents. Among different options, calcium phosphate biomaterials are found to be attractive choices, due to their excellent biocompatibility, customisable bioactivity and biodegradability. Several approaches have been established to enhance this material ability to be loaded with a therapeutic agent, in order to obtain an in situ controlled release that meets the clinical needs. This article reviews the most important factors influencing on both drug loading and release capacity of porous calcium phosphate bone substitutes. Characteristics of the carrier, drug/carrier interactions, experimental conditions of drug loading and evaluation of drug delivery are considered successively.

Hydroxyapatite microporous bioceramics as vancomycin reservoir: Antibacterial efficiency and biocompatibility investigation.

AbstarctInfections after bone reconstructive surgery are a real therapeutic and economic issue for the modern health care system. As the pathogen (most often Staphylococcus aureus) is able to develop a biofilm inside the bone, local delivery of antibiotics is of interest since high drug concentrations would be delivered directly at the target place. In this context, this study evaluated a porous hydroxyapatite implant as biocompatible bone substitute and vancomycin-delivery system to prevent post-operative infections. A simple method of impregnation with optimised conditions insured a high antibiotic loading (up to 2.3 ± 0.3 mg/m2), with a complete in vitro release obtained within 1-5 days. Additionally, the bacteriostatic and bactericidal effects of vancomycin were retained after loading on hydroxyapatite, as demonstrated after challenge with a Staphylococcus aureus strain. Regarding the biocompatibility, a wound healing assay of pre-osteoblastic MC3T3-E1 cells exposed to various concentrations of vancomycin revealed a dose-dependent reduction in cell migration for antibiotic concentrations higher than 1 mg/mL. Meanwhile, cells were able to proliferate normally on vancomycin-loaded scaffolds, although cell initial adhesion was seriously impaired for scaffolds loaded with 2.3 mg/m2 Loaded scaffolds could be stored up to three months at room temperature without any degradation of the antibiotic. Together, these results demonstrate the efficacy of these hydroxyapatite bone substitutes for local delivery of vancomycin in the context of bone infection.

In Situ Microparticles Loaded with S-Nitrosoglutathione Protect from Stroke.

Treatment of stroke, especially during the first hours or days, is still lacking. S-nitrosoglutathione (GSNO), a cerebroprotective agent with short life time, may help if administered early with a sustain delivery while avoiding intensive reduction in blood pressure. We developed in situ forming implants (biocompatible biodegradable copolymer) and microparticles (same polymer and solvent emulsified with an external oily phase) of GSNO to lengthen its effects and allow cerebroprotection after a single subcutaneous administration to Wistar rats. Arterial pressure was recorded for 3 days (telemetry, n = 14), whole-blood platelet aggregation up to 13 days (aggregometry, n = 58), and neurological score, cerebral infarct size and edema volume for 2 days after obstruction of the middle cerebral artery by autologous blood clots (n = 30). GSNO-loaded formulations (30 mg/kg) induced a slighter and longer hypotension (-10 vs. -56 ± 6 mmHg mean arterial pressure, 18 h vs. 40 min) than free GSNO at the same dose. The change in pulse pressure (-50%) lasted even up to 42 h for microparticles. GSNO-loaded formulations (30 mg/kg) prevented the transient 24 h hyper-aggregability observed with free GSNO and 7.5 mg/kg-loaded formulations. When injected 2 h after stroke, GSNO-loaded microparticles (30 mg/kg) reduced neurological score at 24 (-62%) and 48 h (-75%) vs. empty microparticles and free GSNO 7.5 mg/kg and, compared to free GSNO, divided infarct size by 10 and edema volume by 8 at 48 h. Corresponding implants reduced infarct size and edema volume by 2.5 to 3 times. The longer (at least 2 days) but slight effects on arterial pressures show sustained delivery of GSNO-loaded formulations (30 mg/kg), which prevent transient platelet hyper-responsiveness and afford cerebroprotection against the consequences of stroke. In conclusion, in situ GSNO-loaded formulations are promising candidates for the treatment of stroke.

Nitric oxide-eluting scaffolds and their interaction with smooth muscle cells in vitro.

Fabrication of scaffolds loaded with nitric oxide (NO) donors (S-nitrosoglutathione, GSNO, and isosorbide mononitrate, ISMN) with suitable cell compatibility and optimized properties for tissue-engineering applications is reported using "in situ" technique. Based on FDA-approved polymer, solvent and dosage forms, this gentle process allowed the incorporation of the GSNO labile drug into scaffolds made of either poly(lactide-co-glycolide) (PLGA) or PLGA/poly(ɛ-caprolactone) (PCL) blend. During scaffolds manufacturing process including washing cycles, NO donors were leached from scaffolds. However, GSNO and ISMN concentrations in the last washing medium (10(-7) M and 10(-4) M, respectively) were in the range of cell suitability for tissue engineering. Further morphological analyses indicated that smoother surfaces with fewer but bigger pores (compatible with cell penetration and ingrowth) were obtained with PLGA in comparison with PLGA/PCL scaffolds. Among all tested matrices, only unloaded PLGA and GSNO-loaded PLGA/PCL exhibited intermediate cell anchorage, with mitochondrial activity close to the control and an increase in protein content, a prognostic for scaffold cell colonization, defining them as promising candidates. Deeper analyses of these two scaffolds looking at intracellular redox balance through reactive oxygen species production, glutathione, S-nitrosothiols, and nitrite ions content exhibited GSNO-loaded PLGA/PCL as the best of all tested 3D scaffolds for tissue engineering.

PLGA in situ implants formed by phase inversion: critical physicochemical parameters to modulate drug release.

In situ forming implants (ISI) based on phase separation by solvent exchange represent an attractive alternative to conventional preformed implants and microparticles for parenteral applications. They are indeed easier to manufacture and their administration does not require surgery, therefore improving patient compliance. They consist of polymeric solutions precipitating at the site of injection and thus forming a drug eluting depot. Drug release from ISI is typically divided into three phases: burst during precipitation of the depot, diffusion of drug through the polymeric matrix and finally drug release by system degradation. This review gives a comprehensive overview on (i) the theoretical bases of these three phases, (ii) the parameters influencing them and (iii) the remaining drawbacks which have to be addressed to enlarge their commercial opportunities. Indeed, although some of them are already commercialized, ISI still suffer from limitations: mainly lack of reproducibility in depot shape, burst during solidification and potential toxicity. Nevertheless, depending on the targeted therapeutic application, these shortcomings may be transformed into advantages. As a result, keys are given in order to tailor these formulations in view of the desired application so that ISI could gain further clinical importance in the following years.

Are in situ formulations the keys for the therapeutic future of S-nitrosothiols?

S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) were formulated into in situ forming implants (ISI) and microparticles (ISM) using PLGA and either N-methyl-2-pyrrolidone (NMP) or triacetin. Physicochemical characterization was carried out, including the study of matrix structure and degradation. A strong correlation between drug hydrophobicity and the in vitro release profiles was observed: whatever the formulation, GSNO and SNAP were completely released after ca. 1 day and 1 week, respectively. Then, selected formulations (i.e., SNAP-loaded NMP formulations) demonstrated the ability to sustain the vasodilation effect of SNAP, as shown by monitoring the arterial pressure (telemetry) of Wistar rats after subcutaneous injection. Both ISI and ISM injections resulted in a 3-fold extended decrease in pulse arterial pressure compared with the unloaded drug, without significant decrease in the mean arterial pressure. Hence, the results emphasize the suitability of these formulations as drug delivery systems for S-nitrosothiols, widening their therapeutic potential.

S-nitrosation/denitrosation in cardiovascular pathologies: facts and concepts for the rational design of S-nitrosothiols.

Nitric oxide (•NO) is a physiological mediator of vasorelaxation constitutively synthesized by endothelial nitric oxide synthase. Because •NO has a short half-life, it is stored by proteins through S-nitrosation reactions. S-nitrosation was recently defined as a post-translational modification of proteins for cellular signalling, as important as glycosylation and phosphorylation. Disulfide forming/ isomerizing enzymes like thioredoxin (Trx), protein disulfide isomerase (PDI), which are chaperone proteins, are implicated into transnitrosation reactions, which are the transfer of •NO from one cysteine residue to another one. Furthermore, Trx has been shown to denitrosate S-nitrosoproteins depending on its redox status. S-nitrosation of Trx on Cys residues apart from active site, under nitrosative or oxidative stresses, enhances its activity, thereby reducing intracellular reactive oxygen species. Trx and PDI have therefore an essential role for cell signalling control which leads, among other actions, to cardio and vasculo-protection. The diminution of either •NO synthesis or bioavailability is implicated into a large number of cardiovascular pathologies associated to hypoxia or vasoconstriction like, endothelial dysfunction, arterial hypertension and atherosclerosis. In order to mimic the physiological storage of •NO as S-nitrosothiols, the development of •NO donors should be based on the covalent S-NO bond. The chemical stabilisation of the S-NO bond and protection against enzymatically active proteins such as PDI//Trx are major points for the design of stable compounds. S-nitrosothiols entrapment in innovative formulations (films, gels, microparticles, nanoparticles) is an emerging field in order to stabilise and protect them, and to deliver •NO under a sustained release at the targeted site.

Induction of base damages representing a high risk site for double-strand DNA break formation in genomic DNA by exposure of cells to DNA damaging agents.

DNA repair is known as a defense mechanism against genotoxic insults. However, the most lethal type of DNA damages, double-strand DNA breaks (DSBs), can be produced by DNA repair. We have previously demonstrated that when long patch base excision repair attempts to repair a synthetic substrate containing two uracils, the repair produces DSBs (Vispe, S. and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392 and Vispe, S., Ho, E. L., Yung, T. M., and Satoh, M. S. (2003) J. Biol. Chem. 278, 35279-35285). In this synthetic substrate, the two uracils are located on the opposite DNA strands (separated by an intervening sequence stable at 37 degrees C) and represent a high risk site for DSB formation. It is not clear, however, whether similar high risk sites are also induced in genomic DNA by exposure to DNA damaging agents. Thus, to investigate the mechanisms of DSB formation, we have modified the DSB formation assay developed previously and demonstrated that high risk sites for DSB formation are indeed generated in genomic DNA by exposure of cells to alkylating agents. In fact, genomic DNA containing alkylated base damages, which could represent high risk sites, are converted into DSBs by enzymes present in extracts prepared from cells derived from clinically normal individuals. Furthermore, DSBs are also produced by extracts from cells derived from ataxia-telangiectasia patients who show cancer proneness due to an impaired response to DSBs. These results suggest the presence of a novel link between base damage formation and DSBs and between long patch base excision repair and human diseases that occur due to an impaired response to DSB.

15-Hydroxyprostaglandin dehydrogenase in the bovine endometrium during the oestrous cycle and early pregnancy.

Prostaglandins (PG) are primary regulators of reproductive function. In ruminants, the relative production of PGE2 and PGF2alpha determines the return to a new oestrous cycle or to the establishment of pregnancy in response to a viable embryo. PG action depends on biosynthesis, transport and interaction with their receptors, which are all expressed differentially during the oestrous cycle. PGs are, however, local mediators and thus the onsite degradation by enzymes such as 15-hydroxyprostaglandin dehydrogenase (HPGD), also known as 15-PGDH, is another factor to consider in the regulation of physiological action. Little information is available on PG catabolism in the endometrium during the oestrous cycle or early pregnancy. The purpose of this study was to clone the bovine 15-PGDH, produce the recombinant protein and generate a specific antibody to study its activity and its expression in the endometrium during the oestrous cycle. We have found that the bovine 15-PGDH is highly homologous to the rat and human isoforms. 15-PGDH is localized principally in the glandular epithelium and to a lesser extent in stromal and luminal epithelial cells. The enzyme expression is regulated during the oestrous cycle and it reaches its maximal level on days 16-18. Transient expression is observed in luminal epithelial and trophoblast cells on day 21 of pregnancy. The mRNA is expressed at a constant high level throughout the cycle. The activity of the recombinant 15-PGDH was also tested and was found comparable for PGF2alpha and PGE2. These data suggest that 15-PGDH contributes to the tight regulation of PG action in the endometrium especially at the critical period of recognition of pregnancy.

Poly(ADP-ribose) polymerase-1 is a negative regulator of HIV-1 transcription through competitive binding to TAR RNA with Tat.positive transcription elongation factor b (p-TEFb) complex.

Human immunodeficiency virus, type 1 (HIV-1) transcription is regulated by a virus-encoded protein, Tat, which forms a complex with a host cellular factor, positive transcription elongation factor b (P-TEFb). When this complex binds to TAR RNA synthesized from the HIV-1 long terminal repeat promoter element, transcription is trans-activated. In this study we showed that, in host cells, HIV-1 transcription is negatively regulated by competition of poly(ADP-ribose) polymerase-1 (PARP-1) with Tat.P-TEFb for binding to TAR RNA. PARP-1, which has a high affinity for TAR RNA (K(D) = 1.35 x 10(-10) M), binds to the loop region of TAR RNA and displaces Tat or Tat.P-TEFb from the RNA. In vitro transcription assays showed that this displacement leads to suppression of Tat-mediated trans-activation of transcription. Furthermore in vivo expression of luciferase or destabilized enhanced green fluorescent protein genes under the control of the HIV-1 long terminal repeat promoter was suppressed by PARP-1. Thus, these results suggest that PARP-1 acts as a negative regulator of HIV-1 transcription through competitive binding with Tat or the Tat.P-TEFb complex to TAR RNA.

Camptothecin-sensitive relaxation of supercoiled DNA by the topoisomerase I-like activity associated with poly(ADP-ribose) polymerase-1.

Poly(ADP-ribose) polymerase-1 is a highly abundant nuclear enzyme implicated in transcription, DNA replication, and DNA repair through binding of nascent RNA and interactions with various factors. We found that purified fractions of recombinant human poly(ADP-ribose) polymerase-1 expressed in Escherichia coli possess yet another activity, a Mg(2+)-dependent DNA supercoil relaxation activity. Cleavage of recombinant poly(ADP-ribose) polymerase-1 by caspase-3, an apoptotic protease, reduced this activity, as did the removal of either of the two zinc finger motifs located in the N-terminal DNA-binding domain of poly(ADP-ribose) polymerase-1. In addition, this activity was separated from E. coli topoisomerase I by gel-filtration column chromatography, suggesting that this activity is specifically associated with poly(ADP-ribose) polymerase-1. Because this relaxation activity did not require ATP and was resistant to VP16, a topoisomerase II inhibitor, this activity is closer to that of topoisomerase I. However, the supercoiled DNA relaxation activity associated with poly(ADP-ribose) polymerase-1 is distinct from that of human or E. coli topoisomerase I, as this activity could not completely remove superhelical tensions from plasmid DNA. Thus, we referred to this activity as topoisomerase I-like activity. This Mg(2+)-dependent DNA supercoil relaxation activity was found to be sensitive to camptothecin, a mammalian topoisomerase I inhibitor.