[Patient support in pharmaceutical practice: Update on formalised care plans]. / L'accompagnement du patient dans la pratique pharmaceutique : point sur les dispositifs formalisés.

The evolution of patient management has led the pharmacist to change gear and get closer to the patient. To better ensure these missions, several educational and support programs have emerged: "Advice", "consultations" and "pharmaceutical interviews", "shared medication report" or "therapeutic patient education", all these programs are intended "guarantee the best conditions for initiation, monitoring and compliance as well as evaluation of treatment", taking into account the wishes and needs of patients. Although these programs have similarities, there are significant differences (i.e. regulatory, functional, organisational, educational). The aim here is to clarify the various support programs in order to better know their fields of application and put them into practice.

[Design and assessment of a outpatient living with PICC-line support program: A pilot study]. / Étude pilote d'évaluation d'un programme d'accompagnement pour les patients quittant l'hôpital avec un PICC-line.

The peripheral insertion central catheter (PICC-Line) is indicated for long term intravenous medication administration. Some adverse events (AE) might occur, especially for patients after hospital discharge. Therefore, patient empowerment about the side effects and precaution for use is essential to prevent potential patient harm. A multidisciplinary working group met and designed support program for outpatient living with PICC-line. Pharmacy consultations (PC) were proposed to patient before and after PICC-line insertion. A strip cartoon and card game were created to facilitate patient education. The aim of the study was to assess the comprehension of patient then secondarily to follow up AE awareness. During 10 months, 30 patients of mean age 65.9±14 years were included. Thirty-sixPICC-Line were installed and followed on 1659days of catheterization. 4, 9 and 13patients received respectively no, at least one and two PCs before discharge from the hospital. Although the differences were not statistically significant, comprehension tends to improve when patients benefit from both PCs especially when it concerns complications. Twenty-fiveambulatory AEs were recorded including 9infections or suspicion of infection, 2 thrombosis and 2 displacements of PICC-line. Among the patients who had no PC, four experienced delayed care. In comparison, it occurred in only one patient in the group who received at least one PC after PICC-line insertion. Further studies are warranted to confirm this trend.

Efficacy of anakinra for various types of crystal-induced arthritis in complex hospitalized patients: a case series and review of the literature.

BACKGROUND: There are few data on anakinra use after failure of conventional medications for crystal-induced peripheral arthritis and/or crowned dens syndrome among complex hospitalized patients. METHODS: We retrospectively analyzed the outcome of six patients affected with subacute crystal-induced arthritis who had received anakinra in second or third line therapy, including three patients with crowned dens syndrome and three others with gouty arthritis. Patients' comorbidities, reasons for anakinra use and associated drugs, and outcomes were recorded. RESULTS: All patients presented with elevated inflammatory syndrome, systemic symptoms with poly/oligoarthritis. Except for absolute contraindications, all patients were previously treated with full or decreased dose of NSAID, colchicine, and/or glucocorticoids, with unsatisfactory response. All three gouty patients exhibited complete responses in all acute involvements under anakinra within 3 to 5 days, including one of them who needed the reintroduction of colchicine treatment that was previously unsuccessful. Crowned dens syndrome patients, including two with pseudogout and one with subacute hydroxyapatite deposition disease, needed 9 to 11 days to achieve complete response. Tolerance to anakinra was good. CONCLUSION: In case series of complex hospitalized patients, anakinra showed good activity in crowned dens syndrome and associated crystal-induced peripheral arthritis, with longer treatment duration than in gouty arthritis.

Subtelomeric DNA hypomethylation is not required for telomeric sister chromatid exchanges in ALT cells.

Most human tumor cells acquire immortality by activating the expression of telomerase, a ribonucleoprotein that maintains stable telomere lengths at chromosome ends throughout cell divisions. Other tumors use an alternative mechanism of telomere lengthening (ALT), characterized by high frequencies of telomeric sister chromatid exchanges (T-SCEs). Mechanisms of ALT activation are still poorly understood, but recent studies suggest that DNA hypomethylation of chromosome ends might contribute to the process by facilitating T-SCEs. Here, we show that ALT/T-SCE(high) tumor cells display low DNA-methylation levels at the D4Z4 and DNF92 subtelomeric sequences. Surprisingly, however, the same sequences retained high methylation levels in ALT/T-SCE(high) SV40-immortalized fibroblasts. Moreover, T-SCE rates were efficiently reduced by ectopic expression of active telomerase in ALT tumor cells, even though subtelomeric sequences remained hypomethylated. We also show that hypomethylation of subtelomeric sequences in ALT tumor cells is correlated with genome-wide hypomethylation of Alu repeats and pericentromeric Sat2 DNA sequences. Overall, this study suggests that, although subtelomeric DNA hypomethylation is often coincident with the ALT process in human tumor cells, it is not required for T-SCE.

Chemosensitization of fluconazole resistance in Saccharomyces cerevisiae and pathogenic fungi by a D-octapeptide derivative.

Hyperexpression of the Saccharomyces cerevisiae multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the pdr1-3 mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5(+)) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 microg ml(-1)), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic PDR5-null mutant (FLC MIC, 0.94 microg ml(-1)). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer D-octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted D-octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC(50)], 4 microM) which chemosensitized AD/PDR5(+) to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC(50), 30 microM) and Cdr2p (IC(50), 2 microM), and chemosensitized clinical isolates of pathogenic Candida species and S. cerevisiae strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the C. albicans major facilitator superfamily-type drug transporter Ben(R)p, or the FLC drug target lanosterol 14 alpha-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC(50), 1 microM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.

Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters.

Analysis of the transport functions of individual Candida albicans plasma membrane drug efflux pumps is hampered by the multitude of endogenous transporters. We have stably expressed C. albicans Cdr1p, the major pump implicated in multiple-drug-resistance phenotypes, from the genomic PDR5 locus in a Saccharomyces cerevisiae mutant (AD1-8u(-)) from which seven major transporters of the ATP-binding cassette (ABC) family have been deleted. High-level expression of Cdr1p, under the control of the S. cerevisiae PDR5 promoter and driven by S. cerevisiae Pdr1p transcriptional regulator mutation pdr1-3, was demonstrated by increased levels of mRNA transcription, increased levels of nucleoside triphosphatase activity, and immunodetection in plasma membrane fractions. S. cerevisiae AD1-8u(-) was hypersensitive to azole antifungals (the MICs at which 80% of cells were inhibited [MIC(80)s] were 0.625 microg/ml for fluconazole, <0.016 microg/ml for ketoconazole, and <0.016 microg/ml for itraconazole), whereas the strain (AD1002) that overexpressed C. albicans Cdr1p was resistant to azoles (MIC(80)s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 microg/ml, respectively). Drug resistance correlated with energy-dependent drug efflux. AD1002 demonstrated resistance to a variety of structurally unrelated chemicals which are potential drug pump substrates. The controlled overexpression of C. albicans Cdr1p in an S. cerevisiae background deficient in other pumps allows the functional analysis of pumping specificity and mechanisms of a major ABC transporter involved in drug efflux from an important human pathogen.

In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis.

We investigated the in vivo localisation of fission yeast cyclin-dependent kinase cdc2p during mitosis and meiosis. Fusion to yellow fluorescent protein (YFP) revealed that cdc2-YFP is present in the cytoplasm at all stages of the cell cycle. Nuclear cdc2-YFP fluorescence oscillates with that of cdc13-YFP cyclin. At G1/S, at least one of cdc13p, cig1p or cig2p B-type cyclins is required for the accumulation of cdc2-YFP into the nucleus. Cdc2-YFP and cdc13-YFP are highly enriched on the spindle pole body of cells in late G2 or arrested at S phase. Both accumulate on the spindle pole bodies and the spindle in prophase and metaphase independently of the microtubule-associated protein dis1p. In anaphase, the cdc2p/cdc13p complex leaves the spindle prior to sister chromatid separation, and cdc13-YFP is enriched at the nuclear periphery before fluorescence disappears. If cdc13p cannot be recognized by the anaphase-promoting complex, cdc2-YFP and cdc13-YFP remain associated with the spindle. In mating cells, cdc2-YFP enters the nucleus as soon as the cells undergo fusion. During karyogamy and meiotic prophase, cdc2-YFP is highly enriched on the centromeres. In meiosis I, association of cdc2-YFP with the spindle and the spindle pole bodies shows differences to mitotic cells, suggesting different mechanisms of spindle formation. This study suggests that changes in cdc2p localisation are important for both mitosis and meiosis regulation.

The pleitropic drug ABC transporters from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genome contains 16 genes encoding full-size ABC transporters. Each comprises two nucleotide binding folds (NBF) alternating with transmembrane domains (TM). We have studied in detail three plasma membrane multidrug exporters: Pdr5p (TC3.A.1.205.1) and Snq2p (TC3.A.1.205.2) which share NBF-TM-NBF-TM topology as well as Yor1p (TC3.A.1.208.3) which exhibits the reciprocal TM-NBF-TM-NBF topology. The substrate specificity of Pdr5p, Snq2p and Yor1p are largely, but not totally, overlapping as shown by screening the growth inhibition by 349 toxic compounds of combinatorial deletants of these three ABC genes. Multiple deletion of 7 ABC genes (YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11 and PDR15) and of two transcription activation factors (PDR1 and PDR3) renders the cell from 2 to 200 times more sensitive to numerous toxic coumpounds including antifungals used in agriculture or medicine. The use of the pdr1-3 activating mutation and when necessary of the PDR5 promoter in appropriate multideleted hosts allow high levels of expression of Pdr5p, Snq2p or Yor1 p. These overexpressed proteins exhibit ATPase activity in vitro and confer considerable multiple drug resistance in vivo. The latter property can be used for screening specific inhibitors of fungal and other ABC transporters.

Prenyl-flavonoids as potent inhibitors of the Pdr5p multidrug ABC transporter from Saccharomyces cerevisiae.

The Pdr5p multidrug ABC ("ATP-binding cassette) transporter was highly overexpressed in plasma membranes from a yeast strain exhibiting both pdr1-3 gain-of-function mutation in the transcription factor-encoding gene PDR1 and disruption of genes encoding other plasma membrane ABC transporters. Solubilized and purified Pdr5p displayed a tryptophan-characteristic intrinsic fluorescence, whose quenching was used to monitor interactions with substrates and effectors. The transporter exhibited a magnesium-dependent binding affinity for ATP and its fluorescent analogue 2'(3')-N-methylanthraniloyl-ATP, producing a marked fluorescence resonance-energy transfer. It also bound a series of known drug substrates and modulators. Interestingly, yeast Pdr5p interacted with flavonoids recently found to bind to cancer cell P-glycoprotein and to the protozoan parasite multidrug transporter. The extent of high-affinity binding of prenyl-flavonoids to purified Pdr5p was correlated to their efficiency to inhibit energy-dependent quenching of rhodamine 6G fluorescence catalyzed by Pdr5p-enriched plasma membranes. The hydrophobic flavonoid derivative 6-(3, 3-dimethylallyl)galangin was the most efficient, with a K(i) of 0.18 microM for competitive inhibition of the MgATP-dependent quenching of rhodamine 6G fluorescence. In contrast, inhibition of either ATP or UTP hydrolysis occurred at much higher concentrations and appeared to be noncompetitive. Prenyl-flavonoids therefore behave as potent inhibitors of drug binding to the yeast Pdr5p ABC transporter.

Casein kinase I-dependent phosphorylation and stability of the yeast multidrug transporter Pdr5p.

The pleiotropic drug resistance protein, Pdr5p, is an ATP-binding cassette transporter of the plasma membrane of Saccharomyces cerevisiae. Overexpression of Pdr5p results in increased cell resistance to a variety of cytotoxic compounds, a phenotype reminiscent of the multiple drug resistance seen in tumor cells. Pdr5p and two other yeast ATP-binding cassette transporters, Snq2p and Yor1p, were found to be phosphorylated on serine residues in vitro. Mutations in the plasma membrane-bound casein kinase I isoforms, Yck1p and Yck2p, abolished Pdr5p phosphorylation and modified the multiple drug resistance profile. We showed Pdr5p to be ubiquitylated when overexpressed. However, instability of Pdr5p was only seen in Yck1p- and Yck2p-deficient strains, in which it was degraded in the vacuole via a Pep4p-dependent mechanism. Our results suggest that casein kinase I activity is required for membrane trafficking of Pdr5p to the cell surface. In the absence of functional Yck1p and Yck2p, Pdr5p is transported to the vacuole for degradation.

ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p.

The Saccharomyces cerevisiae genome encodes 15 full-size ATP binding cassette transporters (ABC), of which PDR5, SNQ2, and YOR1 are known to be regulated by the transcription factors Pdr1p and Pdr3p (pleiotropic drug resistance). We have identified two new ABC transporter-encoding genes, PDR10 and PDR15, which were up-regulated by the PDR1-3 mutation. These genes, as well as four other ABC transporter-encoding genes, were deleted in order to study the properties of Yor1p. The PDR1-3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins. Overexpressed Yor1p was photolabeled by [gamma-32P]2', 3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP (K0.5 = 45 microM) and inhibited by ATP (KD = 0.3 mM) in plasma membranes. Solubilization and partial purification on sucrose gradient allowed to detect significant Yor1p ATP hydrolysis activity (approximately 100 nmol of Pi.min-1.mg-1). This activity was phospholipid-dependent and sensitive to low concentrations of vanadate (I50 = 0.3 microM) and oligomycin (I50 = 8.5 microg/ml). In vivo, we observed a correlation between the amount of Yor1p in the plasma membrane and the level of resistance to oligomycin. We also demonstrated that Yor1p drives an energy-dependent, proton uncoupler-insensitive, cellular extrusion of rhodamine B. Furthermore, cells lacking both Yor1p and Pdr5p (but not Snq2p) showed increased accumulation of the fluorescent derivative of 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine. Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane. Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes.

Complete inventory of the yeast ABC proteins.

The complete sequence of the yeast genome predicts the existence of 29 proteins belonging to the ubiquitous ATP-binding cassette (ABC) superfamily. Using binary comparison, phylogenetic classification and detection of conserved amino acid residues, the yeast ABC proteins have been classified in a total of six clusters, including ten subclusters of distinct predicted topology and presumed distinct function. Study of the yeast ABC proteins provides insight into the physiological function and biochemical mechanisms of their human homologues, such as those involved in cystic fibrosis, adrenoleukodystrophy, Zellweger syndrome, multidrug resistance and the antiviral activity of interferons.

Identification and characterization of SNQ2, a new multidrug ATP binding cassette transporter of the yeast plasma membrane.

The SNQ2 gene of Saccharomyces cerevisiae, which encodes an ATP binding cassette protein responsible for resistance to the mutagen 4-nitroquinoline oxide, is regulated by the DNA-binding proteins PDR1 and PDR3. In a plasma membrane-enriched fraction from a pdr1 mutant, the SNQ2 protein is found in the 160-kDa over-expressed band, together with PDR5. The SNQ2 protein was solubilized with n-dodecyl beta-D-maltoside from the plasma membranes of a PDR5-deleted strain and separated from the PMA1 H(+/-)ATPase by sucrose gradient centrifugation. The enzyme shows a nucleoside triphosphatase activity that differs biochemically from that of PDR5 (Decottignies, A., Kolaczkowski, M., Balzi, E., and Goffeau, A. (1994) J. Biol. Chem. 269, 12797-12803) and is sensitive to vanadate, erythrosine B, and Triton X-100 but not to oligomycin, which inhibits the PDR5 activity only. Disruption of both PDR5 and SNQ2 in a pdr1 mutant decreases the cell growth rate and reveals the presence of at least two other ATP binding cassette proteins in the 160-kDa overexpressed band that have been identified by amino-terminal microsequencing.

Solubilization and characterization of the overexpressed PDR5 multidrug resistance nucleotide triphosphatase of yeast.

A 160-kDa plasma membrane protein of the yeast Saccharomyces cerevisiae was overexpressed by mutating the PDR1 or the PDR3 transcription factor gene. The protein is the membrane-bound ATP binding cassette transporter PDR5 (Balzi, E., Wang, M., Leterme, S., Van Dyck, L., and Goffeau, A. (1994) J. Biol. Chem. 269, 2206-2214). PDR5 was solubilized with n-dodecyl-beta-D-malto-side and separated from the PMA1 plasma membrane H(+)-ATPase by glycerol gradient centrifugation. The PDR5 protein hydrolyzes nucleoside diphosphates and triphosphates. This activity is sensitive to low concentrations of vanadate, of oligomycin, and of a variety of hydrophobic compounds. Many of these properties liken PDR5 to the purified mammalian P-glycoprotein responsible for multidrug resistance.