Determinants of beta-lactam PK/PD target attainment in critically ill patients: A single center retrospective study.

PURPOSE: We aimed to identify factors associated with achieving target BL plasma concentrations and describe real world data for therapeutic drug monitoring (TDM). METHODS: A retrospective single center study was conducted. We collected data from patients admitted to ICU with at least one BL TDM. We assessed the proportion of patients attaining the recommended plasma concentrations (i.e 100%fT > 4 to 8 MIC). Univariate and multivariate analyses was performed to identify the determinants of BL target attainment. RESULTS: 156 patients were included. At the first dosing, 34% achieved target BL plasma concentrations, 50% were overdosed, and 16% were underdosed. Median time for 1st TDM were 4 (SD = 2.9) days. Multivariate analysis revealed that CKD-EPI estimated glomerular filtration rate (OR = 1.02; CI [1.01; 1.03]; p < 0.0001) and total body weight (OR = 1.03; CI [1.01; 1.04]; p = 0.0048) were the main determinant of BL target attainment. Conversely, Continuous Renal Replacement Therapy (OR = 0.28; CI [0.09; 0.89]; p = 0.0318) and meropenem use (OR = 0.31; CI [0.14; 0.69]; p = 0.0041) were identified as risk factors for overdosing. No factor was associated with underdosing. CONCLUSION: Achieving target BL plasma concentrations remains challenging in ICUs. Identifying predictive factors of BL target attainment would favor implementing rapid dosing optimization strategies in both under and overdosing high risk patients.

MLH1 mediates PARP-dependent cell death in response to the methylating agent N-methyl-N-nitrosourea.

BACKGROUND: Methylating agents such as N-methyl-N-nitrosourea (MNU) can cause cell cycle arrest and death either via caspase-dependent apoptosis or via a poly(ADP-ribose) polymerase (PARP)-dependent form of apoptosis. We wished to investigate the possible role of MLH1 in signalling cell death through PARP. METHODS: Fibroblasts are particularly dependent on a PARP-mediated cell death response to methylating agents. We used hTERT-immortalised normal human fibroblasts (WT) to generate isogenic MLH1-depleted cells, confirmed by quantitative PCR and western blotting. Drug resistance was measured by clonogenic and cell viability assays and effects on the cell cycle by cell sorting. Damage signalling was additionally investigated using immunostaining. RESULTS: MLH1-depleted cells were more resistant to MNU, as expected. Despite having an intact G(2)/M checkpoint, the WT cells did not initially undergo cell cycle arrest but instead triggered cell death directly by PARP overactivation and nuclear translocation of apoptosis-inducing factor (AIF). The MLH1-depleted cells showed defects in this pathway, with decreased staining for phosphorylated H2AX, altered PARP activity and reduced AIF translocation. Inhibitors of PARP, but not of caspases, blocked AIF translocation and greatly decreased short-term cell death in both WT and MLH1-depleted cells. This MLH1-dependent response to MNU was not blocked by inhibitors of ATM/ATR or p53. CONCLUSION: These novel data indicate an important role for MLH1 in signalling PARP-dependent cell death in response to the methylating agent MNU.

DNA loop repair by human cell extracts.

An activity in human cell extracts is described that repairs DNA with loops of five or more unpaired bases. Repair is strand-specific and is directed by a nick located 5' or 3' to the loop. This repair is observed in a colorectal cancer cell line that is devoid of a wild-type hMLH1 gene and is deficient in repair of mismatches. However, a cell line with deletions in both hMSH2 alleles is deficient in repair of both loops and mismatches. Defects in loop repair may be relevant to the repetitive-sequence instability observed in cancers and other hereditary diseases.

[Acute carpal tunnel syndrome caused by a thrombosis of a persistent median artery: 2 case reports]. / Canal carpien aigu : penser à la thrombose de l'artère persistante du nerf médian !

Carpal tunnel syndrome is a common peripheral neuropathy, usually idiopathic or post-traumatic due to the compression of the median nerve. Numbness and paresthesias in the median nerve distribution are the most common symptoms associated with this condition. Persistent median artery is a rare anatomic variation, thrombosis of this additional artery can be responsible for an acute carpal tunnel syndrome, and patients frequently complain about coldness and acute hand swelling. These unusual features must lead clinicians to think of a vascular cause. The diagnosis can be easily confirmed by using ultrasound doppler, but CT-scan and MRI are sometimes helpful. We describe 2 cases of acute carpal tunnel syndrome due to thrombosed persistent median artery, including a case of thromboangiitis obliterans. These thrombosis might also be due to traumatic causes. No guidelines are currently available to help physicians for the management of carpal tunnel syndrome from thrombosed persistent median artery. Antiplatelet therapy, statin, anticoagulant might be helpful, and surgery has sometimes be reported as effective.

Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.

[Molecular pharmacogenetics in hospital laboratories in France: current data and future prospects]. / La pharmacogénétique moléculaire hospitalière en France: données actuelles et perspectives.

Molecular pharmacogenetic units have recently been established in several hospital laboratories in France. The clinical impact of these units is still limited and numerous problems of organizational, ethical, legal, technical, social and economical nature remain to be resolved. However, an increasing number of these units, a rise in their activities and an enlargement of their scope of application are foreseeable in the future. Ultimately, these units would significantly contribute to limit the public health problem caused by interindividual variabilities in drug effects. In view of these prospects, it seems essential that such hospital activity should be quickly recognised by the authorities and the various health sectors in France. It is also essential that the problems that arise from such pharmacogenetic activities should be considered by the authorities and would profit from the organization of a national network and from financial guarantees.

[In vitro fertilization and smoking: use of urinary cotinine and expired air carbon monoxide measurements]. / Fécondation in vitro et tabac: intérêt de la cotininurie et de l'analyseur de monoxyde de carbone expiré.

OBJECTIVE: To study the correlation between expired air carbon (EACO) and urinary cotinine, and to determine the impact of tobacco smoking on in vitro fertilization (IVF) results. PATIENTS AND METHODS: We studied prospectively 221 patients in our ART center from October 2002 to October 2004: 51 active smokers, 85 passive smokers, and 85 non-smokers. Patients were classified into active, passive smokers, or non-smokers, based on a questionnaire. We measured urinary cotinine and EACO on the embryo transfer day and we recorded the IVF parameters. RESULTS: Two hundred and twenty-one patients were included. We observed a 17.2% reduction of estradiolemy (P=0.05), a 1.5% reduction of pregnancies (NS), a 7.8% reduction of infants born alive (NS), a 28.5% reduction of twin pregnancies (P=0.06), as well as a 10% increase of miscarriages (NS) in the active smokers in comparison with non-smokers (the same trends were observed between active and passive smokers). EACO and urinary cotinine were well correlated. There was a negative correlation between estradiolemy and urinary cotinine (R=-0.15, P=0.02). DISCUSSION AND CONCLUSION: Tobacco smoking intensity may be dilatory on IVF results. There is a high correlation between EACO and urinary cotinine. Other larger studies would probably obtain results more statistically significant.

Mutation rate of a microsatellite sequence in normal human fibroblasts.

Dinucleotide repeats, because of their repetitive nature, are prone to frameshift mutations, most likely via a DNA-polymerase slippage mechanism. Mutation rates in microsatellite DNA sequences are high in mismatch repair-defective cells. In normal cells, only estimates of maximal rates of mutation in microsatellites have been possible previously, because of the low sensitivity of screening assays for mutations in endogenous sequences. We have measured the spontaneous mutation rate of a dinucleotide repeat in diploid human foreskin fibroblasts. In our system, the mutation target is a (CA)17 repeat contained within a stably integrated plasmid. The repeat disrupts the reading frame of a neomycin (neo) resistance gene within the plasmid. Cells containing frameshift mutations in the CA repeat that correct the reading frame of the neo gene are selected using the neo analogue G418. This system of measuring microsatellite mutation rates is highly sensitive, because there is a specific target within which mutations can be selected. Fluctuation analysis of cells containing the target DNA yielded mutation rates of <3.1 x 10(-8) to 44.8 x 10(-8) mutations/cell/generation. This is the first report of a direct measurement of a spontaneous mutation rate of a microsatellite sequence in normal human cells.

Role of postreplication repair in transformation of human fibroblasts to anchorage independence.

Cellular capacity for postreplication repair (PRR) and sensitivity to transformation to anchorage independence (AI) were quantified in normal foreskin and xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzo(a)pyrene-diol-epoxide I (BPDE-I). PRR is defined here as a collection of pathways that facilitate the replication of DNA damaged by genotoxic agents. It is recognized biochemically as the process by which nascent DNA grows longer than the average distance between two lesions in the DNA template. PRR refers more directly to the elimination of gaps in the daughter-strand DNA by mechanisms which remain to be determined for human cells, but which may include translesion replication and recombination. PRR was measured in diploid human fibroblasts by analysis of the dose kinetics for inhibition of DNA strand growth in carcinogen-treated cells. Logarithmically growing foreskin fibroblasts (NHF1) displayed D0 values of 4.3 J/m2 and 0.14 microM for the inhibition of DNA synthesis in active replicons by UV and BPDE-I, respectively. XP variant cells (CRL1162) exhibited corresponding D0 values of 1.5 J/m2 and 0.16 microM. The increased sensitivity to inhibition of DNA replication by UV in these XP variant fibroblasts (2.9-fold greater than normal) was mirrored by an enhanced frequency of transformation to AI. XP variant fibroblasts (CRL1162) were 3.2 times more sensitive to transformation to AI by UV than were the normal foreskin fibroblasts. As predicted by the PRR studies, both cell types exhibited similar frequencies of AI colonies induced by BPDE-I. Apparent thresholds were observed for induction of AI by UV (normal fibroblasts, 2.7 J/m2; XP variant fibroblasts, 0.3 J/m2) and BPDE-I (both, 0.05 microM). Doses of UV and BPDE-I above these thresholds produced proportional increases in the inhibition of DNA replication in operating replicons and in the induced frequency of anchorage-independent colonies. At doses of UV and BPDE-I that produced the same degree of inhibition of DNA strand growth, BPDE-I induced a greater number of cells capable of anchorage-independent growth than did UV in both normal and XP variant fibroblasts.

Microsatellite mutation rates are equivalent in normal and telomerase-immortalized human fibroblasts.

Telomerase-expressing human fibroblasts generally have the same properties as normal cells, except that they have an indefinite life span in culture. We have introduced a dinucleotide repeat sequence into the telomerase-expressing hTERT-1604 cell line and characterized the rates and types of frameshift mutations within this microsatellite. These data have been compared with those in diploid fibroblasts with a finite life span. The rates of mutation were found to be similar in the two cell types, indicating that DNA mismatch repair and other cellular processes responsible for maintenance of mutational stability are not disrupted by telomerase immortalization.

Defective postreplication repair in xeroderma pigmentosum variant fibroblasts.

Postreplication repair (PRR) was quantified in normal human fibroblasts and in xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzo[a]pyrene diol epoxide-I (BPDE-I). PRR may be defined as the elimination of discontinuities in the daughter-strand DNA and the replicative bypass of lesions in the DNA template. Pathways of PRR reduce the number of DNA growing points that are blocked at template lesions and increase the rate of growth of nascent DNA on damaged templates. Rates of DNA synthesis and strand growth were measured in solvent- and carcinogen-treated cells by velocity sedimentation analyses of radiolabeled nascent DNA in alkaline sucrose gradients. Logarithmically growing normal fibroblasts displayed D0 values of 6.3 J/m2 and 0.37 microM for the inhibition of DNA synthesis in active replicons by UV and BPDE-I, respectively. Under identical conditions, the XP variant cells exhibited D0 values of 1.5-2.0 J/m2 and 0.27-0.31 microM. Pulse-chase experiments were performed in cells synchronized at the beginning of the S phase. Normal and XP variant cells displayed inhibition of DNA strand growth by UV, with D0 values of 21.6 and 7.0 J/m2, respectively. The D0 values for the inhibition of DNA strand growth by BPDE-I were 0.85 microM for the normal cells and 0.62-0.79 microM for the XP variant cells. The inhibitions of DNA replication by UV and BPDE-I were also analyzed in terms of DNA lesion frequencies. Based on the D0 values for inhibition of DNA replication, we concluded that the XP variant cells express maximally 25-33% of the total PRR activity observed in normal fibroblasts after UV treatment. Conceivably, this deficiency in PRR activity results in the XP variant's increased risk of cancers induced by sunlight, because XP variant cells and normal fibroblasts are equally proficient in excision repair. Both normal and XP variant fibroblasts, however, displayed similar PRR activities in response to BPDE-I treatment.

Fidelity of DNA replication by extracts of normal and malignantly transformed human cells.

To test the hypothesis that a mutator phenotype may be associated with carcinogenesis (L. A. Loeb, Cancer Res., 51: 3074-3079, 1991), we have compared the fidelity of double-stranded DNA replication and the efficiency of mismatch repair in extracts from normal diploid and malignantly transformed human cells. Included was a diploid fibroblast strain and its transformed derivative, as well as a second diploid fibroblast strain and HeLa cells. The fidelity of DNA replication by cytoplasmic extracts in the presence of simian virus 40 large tumor antigen (SV40 T-antigen) was measured using a forward mutagenesis assay. The replicated DNA consisted of double-stranded M13 mp2 DNA containing the SV40 origin of replication and the lacZ alpha complementation gene as a target sequence for scoring mutations. T-antigen-dependent replication was detected in all cell extracts, with those from transformed cells having the greatest activity. No differences in replication fidelity were detected between normal and transformed cell extracts. Using a heteroduplex containing a G.G mispair, we also detected mismatch repair activity in the cell extracts, including efficient repair in extracts from malignantly transformed cells. While these data do not eliminate the possibility that a mutator phenotype may be associated with carcinogenesis, they do suggest that genetic instability associated with transformation does not involve reduced fidelity of replication of undamaged DNA or reduced mismatch repair efficiency.

Microsatellite instability, mismatch repair deficiency, and genetic defects in human cancer cell lines.

The instability of short repetitive sequences in tumor DNA can result from defective repair of replication errors due to mutations in any of several genes required for mismatch repair. Understanding this repair pathway and how defects lead to cancer is being facilitated by genetic and biochemical studies of tumor cell lines. In the present study, we describe the mismatch repair status of extracts of 22 tumor cell lines derived from several tissue types. Ten were found to be defective in strand-specific mismatch repair, including cell lines from tumors of the colon, ovary, endometrium, and prostate. The repair defects were independent of whether the signal for strand specificity, a nick, was 5' or 3' to the mismatch. All 10 defective cell lines exhibited microsatellite instability. Repair activity was restored to 9 of these 10 extracts by adding a second defective extract made from cell lines having known mutations in either the hMSH2 or hMLH1 genes. Subsequent analyses revealed mutations in hMSH2 (4 lines) and hMLH1 (5 lines) that could explain the observed microsatellite instability and repair defects. Overall, this study strengthens the correlation between microsatellite instability and defective mismatch repair and the suggestion that diminuition in mismatch repair activity is a step in carcinogenesis common to several types of cancer. It also provides an extensive panel of repair-proficient and repair-deficient cell lines for future studies of mismatch repair.

Microsatellite instability in gynecological sarcomas and in hMSH2 mutant uterine sarcoma cell lines defective in mismatch repair activity.

We have examined a panel of gynecological sarcomas for microsatellite instability. The genomic DNA from 11 of 44 sarcomas contained somatic alterations in the lengths of one or more di-, tri-, tetra-, or pentanucleotide microsatellite sequence markers, and 6 of these cases had alterations in two or more markers. In addition, di-, tri-, and tetranucleotide microsatellites were found to be highly unstable in single cell clones of two cell lines derived from a uterine mixed mesodermal tumor. Since such instability is characteristic of cells defective in postreplication mismatch repair, we examined mismatch repair activity in extracts made from these lines. Both extracts were repair deficient, while an extract of another gynecological sarcoma cell line not exhibiting microsatellite instability was repair proficient. The repair deficiency was complemented by a colon tumor cell extract that was defective in the hMLH1 protein but not by an extract defective in hMSH2 protein. This suggested that the defect in the uterine sarcoma line could be in hMSH2. Subsequent analysis of the gene revealed a 2-bp deletion in exon 14, leading to premature truncation of the hMSH2 protein at codon 796 and no detectable wild-type gene present. These data suggest that the microsatellite instability observed in these cell lines, and possibly in a significant number of gynecological sarcomas, is due to defective postreplication mismatch repair. There was no apparent correlation with microsatellite instability and clinical outcome.

DNA repair and replication in human fibroblasts treated with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene .

DNA repair and replication were examined in diploid human fibroblasts after treatment with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 microM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4-6 h after treatment with 0.7 microM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 microM) were found to inhibit replicon initiation by up to 50% within 30-60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1 X 10(7) Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 microM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 microM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.

[Measurement of urinary free cotinine. Comparison with the level of expired air carbon monoxide]. / Dosage de la cotinine urinaire libre. Deuxième partie: comparaison avec le taux de monoxyde de carbone dans l'air expiré.

BACKGROUND AND AIM: Cotinine is a very reliable index for the estimation of active or passive smoking. Sampling from a single urine void is well accepted by smokers who are willing to stop. It is not possible to exclude modification of urine cotinine according to beverage intake. The aim of this study was to determine if urine cotinine concentration must necessarily be adjusted to creatinine or not, by making comparison with expired air carbon monoxide. MATERIAL AND METHODS: Carbon monoxide was measured in 53 smokers coming for the first time in a smoking cessation program. Urine cotinine was measured by HPLC-UV. The cut-off value for abstinence is 8ppm and 0.05 mg/L, repectively. Urine creatinine was determined using the Jaffe reaction. RESULTS: Mean CO level was 18.5 +/- 10.6 ppm and mean urine cotine was 1.45 +/- 0.86 mg/L. Eight smokers had CO 8 ppm. They should be considered as abstinent. However, only one of them had a cotinine under the detection limit. Urine creatinine varied in a large range (0.7 - 35 mmol/L). But, cotinine was only weakly correlated to creatinine (r = 0.279, p = 0.037). There was a highly significant correlation between cotinine and CO (0.649, p = 0.0001). The correlation of cotinine/creatinine versus CO was not significant (r = 0.249, p = 0.072). In order to take into account fluid intake, urine cotinine of each sample was adjusted as if creatinine was equal to the mean (8.3 mmol/L) of the group of subjects. The correlation observed with adjusted or non adjusted cotinine and CO (r = 0.640, p < 0.0001) was the same. CONCLUSION: Urine cotinine from a single void is an accurate index of tobacco smoking at the individual level. There is no need to adjust cotinine concentration, taking into account urine creatinine. Measurement of urine cotinine can be useful to manage smokers who deliberately wish to overcome tobacco dependence, offering the opportunity to provide an adequate level of nicotine substitutive therapy. It is also of peculiar importance to follow-up pregnant women and smokers for whom cessation is required after a clinical event. Finally, absence of cotinine in urine can be used to document abstinence from tobacco products.

[Urinary cotinine and nicotine metabolites measurement]. / Dosage urinaire de la cotinine et des métabolites de la nicotine.

UNLABELLED: According to the recent regulations (Circulaire DGS/DH du 3 avril 2000), tobacco dependence must be determined by the measurement of urine nicotine metabolites. Various assay methods are presently available. They were tested in order to evaluate their analytical performances and to determine how they can be used for the clinical management of smoking cessation. MATERIAL AND METHODS: Urine samples from a single void (n = 97) were obtained from active and abstinent smokers (with or without nicotine substitutive therapy). They were all analyzed by the various methods. Cotinine concentration was measured in six laboratories, using HPLC combined with UV detection according to a standardized procedure (Ann Biol Clin 2002 : 60 : 263-72). Immunoassay methods were also tested and the values obtained from urine samples were compared to urine cotinine measured by HPLC-UV. RESULTS: HPLC-UV: Urinary cotinine varied in a range from undetectable to 4 mg/L. An interlaboratory comparison was performed according to the Valtec procedure (calculation of equation of Deming, chart of differences). There was a good accordance between laboratories. Cotinine concentration was only slightly influenced by fluid intake, as shown by a poorly significant correlation between cotinine and creatinine (r = 0.23, p = 0.05). Homogeneous immunoassays: The two homogeneous immunoassays (Cotinine) from Thermo Electron and Cotinine Enzyme Immunoassay commercialized by Microgenics were highly correlated (r = 0.97). The correlation was not so strong with HPLC-UV (r = 0.86). Firstly, values were found higher with immunoassays because antibodies crossreact with 3-hydroxycotinine. Secondly, the ratio of immunoassays values to HPLC-UV values varied according to urine specimens. Finally, there was a highly significant correlation with urine creatinine (r = 0.40, p = 0.0001), thus indicating the influence of fluid intake. Heterogeneous immunoassay: The kit Metabolites of Nicotine commercialized by DPC France was tested on the analyzer Immulite, using a procedure specifically established for urine. Antibodies revealed a large spectrum of nicotine metabolites. Therefore, the values were much higher than those observed for the same urine samples with homogeneous immunoassays. CONCLUSION: HPLC-UV can be recommended for the measurement of urinary cotinine, as it was shown a good accordance between laboratories. The low detection limit is of interest for the diagnosis of Environmental Tobacco Smoking. Homogeneous immunoassays can be easily used for routine analysis as they can be performed directly on urine specimen. The results must be interpreted according to cut-off values specifically established according to homogeneous or heterogeneous immunoassays. Variability induced by fluid intake must be taken into account. The interest of the heterogeneous immunoassay needs to be confirmed for the diagnosis of Environmental Tobacco Smoking.

Contraction of human colonic circular smooth muscle cells is inhibited by the calcium channel blocker pinaverium bromide.

UNLABELLED: The effects of L-type calcium channel blockers (CCBs) selective for the gastrointestinal tract (pinaverium) or non-selective (nicardipine and diltiazem), were investigated on CCK-, CCh- or KCl-induced contraction of smooth muscle cells (SMC) isolated from the circular muscle layer of normal or of inflamed human colons. In the normal tissue colon, whatever the contractile agent used, CCK-8 (1nM), CCh (1nM) or KCl (20mM), a micromolar concentration of pinaverium significantly inhibited contraction (88.36%, 93.10%, 93.92% inhibition respectively); this effect was concentration-dependent for CCh (IC50 = 0.73 +/- 0.08nM) and for CCK (IC50 = 0.92 +/- 0.12nM). In parallel, both nicardipine and diltiazem inhibit significantly contraction of isolated SMC. In inflamed colons, pinaverium (1 microM) display a significant higher efficacy than diltiazem or nicardipine to reduce cell contraction induced by CCK-8 or by KCl. In addition, RT-PCR experiments were performed to evidence tissue specificity of the L-type calcium channel. They revealed the expression of the messenger of the a-1 subunit L-type calcium channel (binding site of such CCBs), consistent with the expression of the rbC-2 splice variant of the alpha1-C gene. IN CONCLUSION: (i) the inhibition by calcium channel blockers of agonist-induced contractile activity suggest a modulation of SMC contraction upon extracellular calcium via 'L-type' voltage-dependent calcium channel; (ii) this study provides a rationale for the clinical use of pinaverium in colonic motor disoders affecting the contractility of SMC, since it appeared to decrease the contraction even in pathological situation; and (iii) RT-PCR experiments confirms the presence in human colon SMC of the alpha-1 subunit mRNA of calcium channel.

Transgenic plants that express genes including the 3' untranslated region of the turnip yellow mosaic virus (TYMV) genome are partially protected against TYMV infection.

In order to evaluate new possibilities for protecting plants against virus infection by interference with viral replication, two chimeric genes were constructed in which the (+) strand 3'-terminal 100 nucleotides (nt) of the noncoding region of the turnip yellow mosaic virus (TYMV) genome were placed downstream from the sense or antisense cat coding region. The two chimeric genes were then introduced into the genome of rapeseed (Brassica napus) using an Agrobacterium rhizogenes vector system. Plants expressing high levels of either chimeric gene showed partial protection against infection by TYMV RNA or virions. One interesting feature of the protection is that a proportion of the inoculated transgenic plants does not become infected. Protection was overcome when the inoculum concentration was increased. RNA complementary to the initial transcript was detected after infection.

Plant viruses and new perspectives in cross-protection.

Cross-protection in plants is the phenomenon whereby a plant preinoculated with a mild virus strain becomes resistant to subsequent inoculation by a related severe strain. It has been used on a large scale in cases where no resistant plants are available. Although several hypotheses have been proposed to explain the molecular mechanism underlying cross-protection, no single hypothesis can account for all the data obtained. Recently, a phenomenon akin to cross-protection has been achieved in transformed plants harboring the cDNA of a part of a viral RNA genome. These results obtained by genetic engineering raise new hopes for obtaining plants resistant to virus infection.