Label-free and reagentless electrochemical detection of microRNAs using a conducting polymer nanostructured by carbon nanotubes: application to prostate cancer biomarker miR-141.

In this paper, a label-free and reagentless microRNA sensor based on an interpenetrated network of carbon nanotubes and electroactive polymer is described. The nanostructured polymer film presents very well-defined electroactivity in neutral aqueous medium in the cathodic potential domain from the quinone group embedded in the polymer backbone. Addition of microRNA miR-141 target (prostate cancer biomarker) gives a "signal-on" response, i.e. a current increase due to enhancement of the polymer electroactivity. On the contrary, non-complementary miRNAs such as miR-103 and miR-29b-1 do not lead to any significant current change. A very low detection limit of ca. 8 fM is achieved with this sensor.

Label-free and reagentless electrochemical detection of PCR fragments using self-assembled quinone derivative monolayer: application to Mycobacterium tuberculosis.

We report a signal-on, label-free and reagentless electrochemical DNA biosensor, based on a mixed self-assembled monolayer of thiolated hydroxynaphthoquinone and thiolated oligonucleotide. Electrochemical changes resulting from hybridization were evidenced with oligonucleotide targets (as models), as well as with polymerase chain reaction (PCR) products related to different lineages of Mycobacterium tuberculosis strains. With pure oligonucleotides, this system achieves high sensitivity (∼300 pM of DNA target, i.e. 30 fmol in a 100 µL sample) and excellent selectivity, allowing to detect a single mismatch on a sequence of 20 bases. With PCR products, current changes are specific to the bacterial strain from which the PCR fragment is produced. In addition, the sensor response is of the signal-on type, giving a positive signal change upon hybridization, and therefore does not suffer from false positive responses due to non-specific adsorption of DNA.

A label-free electrochemical immunosensor for direct, signal-on and sensitive pesticide detection.

A new electropolymerizable monomer, [N-(6-(4-hydroxy-6-isopropylamino-1,3,5-triazin-2-ylamino)hexyl) 5-hydroxy-1,4-naphthoquinone-3-propionamide], has been designed for use in a label-free electrochemical immunosensor when polymerized on an electrode and coupled with a monoclonal anti-atrazine antibody. This monomer contains three functional groups: hydroxyl group for electropolymerization, quinone group for its transduction capability, and hydroxyatrazine as bioreceptor element. Square wave voltammetry shows that the polymer film, poly[N-(6-(4-hydroxy-6-isopropylamino-1,3,5-triazin-2-ylamino)hexyl) 5-hydroxy-1,4-naphthoquinone-3-propionamide], presents negative current change following anti-atrazine antibody complexation and positive current change after atrazine addition in solution. This constitutes a direct, label-free and signal-on electrochemical immunosensor, with a very low detection limit of ca. 1 pM, i.e. 0.2 ng L(-1), one of the lowest reported for such immunosensors. This is far lower than the detection limit required by the European Union directives for drinkable water and food samples (0.1 µg L(-1)). The strategy described has great promise for the development of simple, cost-effective and reagentless on-site environmental monitors.

Design of a new electrogenerated polyquinone film substituted with glutathione. Towards direct electrochemical biosensors.

We developed a method to graft a tripeptide (glutathione) onto 5-hydroxy-1,4-naphthoquinone, an electropolymerizable molecule. The resulting thin conducting polymer presents a well-defined and stable electroactivity in neutral buffered solution, due to the embedded quinone group, and is able to covalently graft amino-modified DNA probe strands. It is shown that the bioelectrode presents positive current change following DNA hybridization. This makes a "signal-on" direct electrochemical DNA sensor. The results were obtained with low target concentration (50nM) and the selectivity is excellent as a single-mismatch sequence can be discriminated from the full-complementary target.

Reagentless amperometric detection of l-lactate on an enzyme-modified conducting copolymer poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-3-thioacetic acid-1,4-naphthoquinone).

We have constructed a reagentless lactate sensor using lactate oxidase (LOD) covalently attached to an electropolymerized copolymer film, poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-3-thioacetic acid-1,4-naphthoquinone), poly(JUG-co-JUGA). Around 10(-12)Mcm(-2) of covalently bound enzymes are immobilized on these films. In aerated medium, the amperometric response versus lactate concentration shows a sensitivity of 350 +/- 50 microAM(-1)cm(-2) for an applied potential of +0.5V versus Ag|AgCl on a film-coated Pt electrode. In deaerated medium, the quinone group, conjugated with the polymer backbone, acts as an immobilized mediator. An amperometric response is observed on film-coated glassy carbon (GC) electrode at a potential as low as -0.1V versus Ag|AgCl, with a sensitivity of 110 +/- 40 microAM(-1)cm(-2).

Direct electrochemical detection of oligonucleotide hybridization on poly(5-hydroxy-1,4-naphthoquinone- co-5-hydroxy-3-thioacetic acid-1,4-naphthoquinone) film.

We describe the construction of a new DNA-modified electrode based on an electroactive film. 5-Hydroxy-1,4-naphthoquinone is coelectrooxidized with 5-hydroxy-3-thioacetic acid-1,4-naphthoquinone to give a copolymer, presenting both electroactive and chemically reactive groups. The carboxylic function acts as a precursor for the covalent grafting of ODN probes while the quinone group acts as the transduction element of hybridization. Electrochemical detection was performed by differential pulse voltammetry in the electroactivity domain of the quinone group (i.e., at very low potentials, 0 to -0.8 V vs SCE). A very clear modification of the redox activity is observed between unmodified and probe-modified films and especially upon addition of target ODN.

Synthesis and antitumor activity of ester-modified analogues of bengamide B.

Bengamide B, a novel sponge-derived marine natural product with broad spectrum antitumor activity, was not suitable for further preclinical development because of its difficult synthesis and very poor water solubility. Bengamide B produced a 31% T/C at its solubility-limited maximum intravenous dose of 33 micromol/kg in MDA-MB-435 breast carcinoma implanted subcutaneously as a xenograft in nude mice. Compound 8a, a bengamide B analogue with three structural changes (t-Bu alkene substituent, unsubstituted lactam nitrogen, and inverted lactam 5'-myristoyloxy group), was as potent as bengamide B in vitro and more efficacious than bengamide B in vivo. A series of ester-modified analogues based on 8a were synthesized and tested in vitro and in vivo (MDA-MB-435). The cyclohexyl- and phenethyl-substituted esters, 8c and 8g, respectively, had in vitro and in vivo activities similar to that of 8a and enhanced water solubility (ca. 1 mg/mL). Consequently, 8c and 8g were tested in the MDA-MB-435 xenograft model at 100 micromol/kg and produced 29% and 57% tumor regression, respectively.

Total syntheses of bengamides B and E.

Total syntheses of the cytotoxic marine natural products bengamides B and E are described. Both bengamides are prepared via amide coupling of a protected polyhydroxylated lactone intermediate 9 with a suitably substituted aminocaprolactam intermediate. Lactone 9 is prepared in five steps from commercially available alpha-D-glucoheptonic gamma-lactone. The key reactions are a selective deprotection of a 1,2-acetonide in the presence of a 1,3-acetonide and an (E)-selective olefination of an unstable aldehyde using a gem-dichromium reagent. The bengamide B lactam intermediate 10 is prepared in seven steps from commercially available (5R)-5-hydroxy-L-lysine (12). The desired S-configuration at the gamma-OH lactam position is established using the Mitsunobu reaction.

Use of the robust sib-pair method to screen for single-locus, multiple-locus, and pleiotropic effects: application to traits related to hypertension.

Robust sib-pair linkage analysis can be used as a screening tool in the search for the potential involvement of single-loci, multiple-loci, and pleiotropic effects of single loci underlying phenotypic variation. Four large families were each ascertained through one adult white male with essential hypertension. The robust sib-pair method was used to screen these families for evidence of linkage between 39 quantitative traits related to hypertension and 25 genetic marker loci. All traits were analyzed on the untransformed, square-root and log-transformed scales. Among other findings, there is a suggestion of linkage between the 6-phosphogluconate dehydrogenase locus on chromosome 1p36 and mean fifth-phase diastolic blood pressure. There may also be linkage between the following markers and traits: the adenylate kinase-1 marker and/or the Lewis blood group marker and the traits height, weight, and biacromial breadth; the glyoxylase I marker and the traits upper-arm circumference and suprailiac skinfold thickness; the ABO blood group and adenylate kinase-1 markers on chromosome 9q34 and the third component of complement marker on chromosome 19p13 and dopamine-beta-hydroxylase; and the P1 blood group and the traits weight and 1-h postload serum glucose level.

Stepwise oligogenic segregation and linkage analysis illustrated with dopamine-beta-hydroxylase activity.

A stepwise oligogenic method is developed that can be used to adjust the phenotype of a quantitative trait for the effects of a previously identified single-locus component. This method assumes that a single-locus component can be adequately identified through the use of segregation and/or linkage analysis under a 1-locus model and that the variation due to that locus can be removed from the phenotype leaving a residual that can be parameterized in terms of an additional single-locus component. Segregation and/or linkage analysis can then be used in an attempt to identify an additional single-locus component in the residual phenotype. This stepwise process can be repeated until no further single-locus effects are identified. The method is illustrated using family data on the specific activity of dopamine-beta-hydroxylase (DBH), which a number of studies have suggested may be due either to the combined effects of single-locus and multifactorial components or to the combined effects of 2 loci.

Is adrenal medulla involved in the antihypertensive effect of nicardipine?

The effects of nicardipine, a dihydropyridine calcium channel blocker, on adrenal medulla were investigated in chloralose-anesthetized dogs. For analysis of the adrenal medullary function, adrenal venous catecholamine rates were determined. Intravenous administration of nicardipine (50 micrograms/kg) induced a marked increase in both adrenal catecholamine rates and heart rate and a simultaneous decrease in blood pressure. This effect on epinephrine and norepinephrine release is probably explained by the involvement of baroreflex mechanisms. After acute splanchnicectomy, nicardipine (50 and 100 micrograms/kg i.v.) failed to modify catecholamine secretion rates from the denervated adrenal medulla during electrical stimulation of the splanchnic nerve at low (2 Hz) and high (5 Hz) frequencies. In conclusion, these results suggest that in vivo a functional dihydropyridine-sensitive calcium channel is not required for calcium entry mechanisms into dog chromaffin cells. Moreover, adrenal medulla is not involved in the antihypertensive action of nicardipine.

Linkage of a gene regulating dopamine-beta-hydroxylase activity and the ABO blood group locus.

Previous studies have presented evidence suggesting that levels of dopamine-beta-hydroxylase (DBH) activity are controlled by a gene linked to the ABO blood group locus. In this study, linkage analyses in four large families of whites and one family of blacks were performed on the untransformed and on the square root--and natural log--transformed DBH activity. In the families of white individuals, the results of both the sib-pair and lod-score linkage analyses strongly indicate that a gene regulating DBH activity is linked to the ABO blood group locus on chromosome 9q (i.e., lod score 5.88 at a recombination fraction of .0). However, the transformation used has a large effect on the maximum lod score and estimated recombination fraction. This putative gene does not appear to be polymorphic in the family of blacks.

Effects of lysine-vasopressin and oxytocin on central cardiovascular control.

1 The cardiovascular effects of intravenous and intracisternal administration of neurohypophysial peptides were compared in dogs anaesthetized with chloralose. 2 Intravenous lysine-vasopressin (0.1 to 100 mu/kg) induced a dose-dependent increase in blood pressure and a decrease in heart rate. In contrast, intracisternal lysine-vasopressin (0.01 to 10 mu/kg induced a dose-related decrease in blood pressure and did not change heart rate. 3 Intracisternal oxytocin (1 and 10 mu/kg) increased blood pressure and did not change heart rate, whereas the same doses injected intravenously were inactive. 4 Pretreatment with guanethidine (15 mg/kg i.v. 24 h beforehand) abolished the hypotensive responses to intracisternal vasopressin but not the pressor action of intravenous vasopressin. 5 The pressor responses to central injections of oxytocin were not modified by guanethidine. 6 Hypotension elicited by intracisternal vasopressin was probably due to a decrease in sympathetic tone whereas the hypertension induced by intracisternal oxytocin was independent of variations in sympathetic tone.

Evidence for the involvement of central alpha 2 mechanisms in intrathoracic volume expansion--induced diuresis: a study with clonidine and propranolol.

Intravenous (10 micrograms/kg) or intracisternal (1 microgram/kg) clonidine inhibited the diuretic response to negative pressure breathing (NPB) and left atrial distension (LAD) in chloralose anesthetized dogs. The drug reduced the induced tachycardia, but not the increase in respiratory rate caused by NPB, and did not change the blood pressure. Propranolol (1 mg/kg i.v.) did not change the NPB-induced diuresis. Intravenous yohimbine (1 mg/kg i.v.) opposed the effects of intravenous or intracisternal clonidine, whereas prazosin (0.05 mg/kg i.v.) had no effect. These results show that the adrenergic receptor implicated in the volumetric control of vasopressin secretion could be of the alpha 2 subtype. This alpha 2 adrenoceptor could be centrally located. Clonidine might therefore be proposed to combat the dehydration observed after long-term weightlessness.

[Demonstration of a central hypotensive action of lysine vasopressin in the dog]. / Mise en évidence d'une action hypotensive centrale de la lysine vasopressine chez le chien.

In chloralose-anaesthetized dogs intravenous infection of lysine-vasopressin (0.01 to 100 mU/kg) induced a dose-dependent increase in blood pressure and a decrease in heart rate. The same dosis injected by central (intracisternal) route induced a constant decrease in blood pressure. These results suggest the involvement of vasopressin in central cardiovascular control and in pathogenesis of some varieties of arterial hypertension.

Blockade by clonidine of negative pressure breathing-induced diuresis in the dog.

The effects of clonidine and dl-propranolol on negative pressure breathing-induced diuresis in chloralose-anaesthetized dog were studied. Clonidine (10 microgram/kg i.v.) suppressed the diuretic response, whereas dl-propranolol (1 mg/kg i.v.) was inactive.

[Influence of metabolic alkalosis on adrenaline hyperglycemia of the dog]. / Influence de l'alcalose métabolique sur l'hyperglycémie adrénalinique du chien

The metabolic alkalosis, induced by the administration bicarbonate, reduces adrenaline hyperglycemia in fasted dog.