Fast determination of propranolol in urine and pharmaceutical preparations by stopped-flow and micellar-stabilized room-temperature phosphorescence: validation of the method

The stopped-flow mixing technique has been used to study the kinetic determination of propranolol by means of micellar-stabilized room-temperature phosphorescence. This mixing system diminishes the time required for the deoxygenation of micellar medium by sodium sulfite, allowing a kinetic curve that levels off within only 7s to be obtained. The phosphorescence enhancers thallium (I) nitrate, sodium dodecyl sulfate, and sodium sulfite were optimized to obtain maximum sensitivity and selectivity. A pH value of 6.54 was selected as adequate for phosphorescence development. The kinetic curves of propranolol phosphorescence were scanned at lambda(ex)=290 nm and lambda(em)=524 nm. The calibration graphs were linear for the concentration range from 25 to 400 ng mL(-1). The phosphorescence lifetime of propranolol is approximately 1210 micros. The detection limit calculated as proposed clayton was 13.53 ng mL(-1) and by applying the error propagation theory, the detection limit was 8.37 ng mL(-1). The repeatability was studied using 10 solutions of 200 ng mL(-1) of propranolol; if error propagation theory is assumed, the relative error is 1.94%. The standard deviation for a replicate sample was 4.0 ng mL(-1). This method was successfully applied to the determination of propranolol in commercial formulations and in urine. Suitable recovery values were obtained.     

Immunoaffinity removal of xenoreactive antibodies using modified dialysis or microfiltration membranes

Hyperacute rejection following xenogeneic transplantation in primates is mediated by naturally occurring IgM antibodies, which are specifically directed to alpha-Galactosyl residues on many nonprimate mammalian cells. Current approaches to remove these anti-alphaGal IgM include plasmapheresis followed by immunoaffinity adsorption on bead columns using synthetic Gal epitopes, which requires two pieces of complex equipment. In this study, we explored the use of immunoaffinity adsorption with hollow fiber microporous or dialysis membranes to which a synthetic alphaGal trisaccharide ligand is bound. Covalent attachment of ligand directly to the surface produced negligible binding, but use of long-chain polyamines as reactive spacers yielded binding densities for anti-alphaGal IgM as high as 89 mg/mL membrane volume in breakthrough curve experiments with microporous nylon membranes having an internal surface area of 4.2 m(2)/mL membrane volume. A crossflow microfilter fabricated from the membranes described in this study and having about 0.4 m(2) luminal surface area would be able to carry out plasma separation and immunoadsorption in a single device with a large excess of binding capacity to ensure that all plasma that filters across the device and is returned to a human patient is essentially free of anti-alphaGal IgM. We conclude that immunoaffinity removal of xenoreactive antibodies using microfiltration hollow fiber membranes is feasible and has potential advantages of efficiency and simplicity for clinical application.     

Preparative purification of B-phycoerythrin from the microalga Porphyridium cruentum by expanded-bed adsorption chromatography

B-Phycoerythrin (B-PE) is a major light-harvesting pigment of microalgae. Due to its high fluorescence efficiency and its intense and unique pink color, it is widely used as a fluorescent probe and analytical reagent as well as being employed as a natural dye in foods and cosmetics. Tedious methodologies for B-PE purification have been published. In this work we present a new, fast, preparative and scaleable two-step chromatographic method for B-PE purification from the red microalga Porphyridium cruentum. Initially, phycobiliproteins were released from the microalga cells by osmotic shock and captured by applying the centrifuged cell suspension to a column containing 74 ml Streamline-DEAE equilibrated with 50 mM acetic acid-sodium acetate buffer, pH 5.5, using expanded-bed adsorption chromatography at an upward flow of 200 cm h(-1). After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and a B-PE-rich solution was eluted with a downward flow of the same 250 mM buffer. In order to obtain pure B-PE, we utilized conventional ion-exchange chromatography with a column of DEAE-cellulose loaded directly with the eluate from Streamline-DEAE and developed using a discontinuous gradient of acetic acid-sodium acetate buffer, pH 5.5. With this new methodology, 66% of B-PE contained in the biomass of the microalgae was recovered, a value significantly higher than those obtained following other methodologies. The B-PE purity was tested using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectroscopic characterization.     

One gene determines maize B chromosome accumulation by preferential fertilisation; another gene(s) determines their meiotic loss

Genotypes of high (H(m)) and low (L(m)) male B transmission rate (B-TR) were obtained. B-TR segregation in the F2 is reported, showing that the H(m) and L(m) lines differ in a single locus we call mBt (male B transmission), controlling B preferential fertilisation in maize. The egg cells control which one of the sperm nuclei is going to fertilise them, mBt(h) egg cells being preferentially fertilised by the sperm nucleus carrying the supernumerary B chromosomes (Bs). It is hypothesised that the mBt gene is involved in the normal fertilisation of maize but the parasitic Bs take advantage of the mBt(h) allele to increase their own transmission. Selection was also carried out when the Bs were transmitted on the female side (H(f) and L(f) lines). The F1 hybrids show that the gene(s) that we call fBt (female B transmission), controlling female B-TR, is located on the A chromosomes acting at diploid level, the fBt(l) allele(s) for low transmission being dominant. This allele causes the loss of Bs at meiosis, which is shown using a specific B molecular probe to determine B presence/absence in microspores of both lines and hybrids. Maize Bs are a nice example of intragenome conflict, because the mBt and fBt loci are a polymorphic system of attack and defence between A and B chromosomes.     

Activity of antioxidant enzymes in response to cadmium in Crotalaria juncea

The aromatic amine, -phenethylamine, was identified in various field-grown leguminous plants by analyses with HPLC, GC, GC-MS and ¹H-NMR. High concentration of -phenethylamine was generally detected only in mature root nodules, but not in other plant organs such as root, stem, leaf, pod and grain. Occurrence was specific to the root nodules formed by Bradyrhizobium infection. Ten of eleven legume crops including soybean [Glycine max (L.) Merr.], pigeon pea [Cajanus cajan (L.) Millsp.], adzuki bean (Vigna angularis), mung bean [V. radiata (L.) Wilczek] and cowpea (V. unguiculata) contained this aromatic amine, but groundnut (Arachis hypogaea L.) also nodulated by Bradyrhizobium sp. did not. Root nodules collected from garden pea (Pisum sativum L.), broad bean (Vicia fava L.), kidney bean (Phaseolus vulgaris L.) and various other herbaceous legumes nodulated by Rhizobium sp., Mesorhizobium sp., Sinorhizobium sp. or Azorhizobium caulinodans, and root-nodulated, woody non-legumes, nodulated by Frankia spp., contained little -phenethylamine.The amount of -phenethylamine in Bradyrhizobium-infected nodules varied with the legume species and their cultivars, and most significantly, with nodule age. In field-grown soybean plants, nodule -phenethylamine attained maximum concentration at the flowering stage and far exceeded that of the major polyamines of soybean nodules, putrescine and spermidine.     

Mitogen-activated protein kinase phosphorylates and targets inducible cAMP early repressor to ubiquitin-mediated destruction

Inducible cAMP early repressor (ICER) is an important mediator of cAMP antiproliferative activity that acts as a putative tumor suppressor gene product. In this study, we examined the regulation of ICER protein by phosphorylation and ubiquitination in human choriocarcinoma JEG-3 and mouse pituitary AtT20 cells. We found that cAMP stabilized ICER protein by inhibiting the mitogen-activated protein kinase (MAPK) cascade. Activation of the MAPK pathway increased ICER phosphorylation. ICER phosphorylation was abrogated by inhibition of the MAPK pathway either by cAMP or directly by the MAPK inhibitor PD098059. The MAPKs extracellular signal-regulated kinases 1 and 2 physically interact with ICER and mediated the phosphorylation of ICER on a critical serine residue (Ser-41). A mutant form of ICER in which Ser-41 was substituted by alanine had a half-life 4-5 h longer than its wild-type counterpart. This alteration in stability was due to the inability of the Ser-41-mutant ICER to be efficiently ubiquitinated and degraded via the ubiquitin-proteasome pathway. These results present a novel cell signaling cross-talk mechanism at the cell nucleus between the MAPK and cAMP pathways, whereby MAPK targets a repressor of the cAMP-dependent gene expression for ubiquitination and proteasomal degradation.