Automated amplified flow immunoassay for cocaine

An amplified flow immunoassay (AFIA) was developed for cocaine, which combines a noncompetitive immunoenzymometric assay (IEMA) with an on-line detection of the enzyme label alkaline phosphatase (ALP) by a substrate-recycling biosensor. In the IEMA, the analyte cocaine first binds to a labeled polyclonal anti-cocaine antibody. Then, the excess labeled antibody is separated on an affinity column that contains a perfusion chromatography carrier modified by immobilized cocaine. The unbound complexes of the analyte cocaine with the ALP-labeled antibody are detected postcolumn. The detector senses phenol produced by ALP from phenyl phosphate. As detector, an amperometric substrate-recycling biosensor was used, which consists of a Clark-type oxygen electrode covered by tyrosinase and pyrroloquinoline quinone-dependent glucose dehydrogenase. The lower limit of detection is 380 pM (38 fmol) for cocaine. The sampling rate is 26/h. Cocaine could be detected from "real samples" with an imprecision of +/- 10% (n = 3) and with a recovery of 49 +/- 3% for various concentrations. AFIA is generally important as a new approach for the fast detection of picomolar concentrations of haptens.     

Effects of four different methods of sampling arterial blood and storage time on gas tensions and shunt calculation in the 100% oxygen test

At the present time, plastic syringes are most commonly used for collecting arterial blood. The oxygen tension of the arterial blood (Pa,O2) in these syringes may fall. We studied the effect of the type of syringe, metabolism, and storage time on the arterial oxygen pressures measured and on the pulmonary shunt calculated. In 10 patients, 2-3 h after aortacoronary bypass surgery, a 100% oxygen test was performed. Four arterial blood gas samples were withdrawn from each patient in random order, two in glass syringes and two in plastic syringes. One glass and one plastic syringe were stored at room temperature (RT), and the others were stored in ice-water (IW). Each sample was analysed as soon as possible, and repeated 15, 30, 60 and 120 min after sampling. The Pa,O2 measurement in blood in the glass syringe in IW measured as soon as possible after sampling was considered the "gold standard". Pulmonary shunt calculations were performed using the results of the various blood gas analyses. Compared with the "gold standard", all of the other methods showed significant deterioration in the Pa,O2 measurement. The effect due to diffusion was 0.05 kPa x min(-1), and that due to metabolism 0.11 kPa x min(-1). The Pa,O2 in the glass syringes stored in IW remained stable with time. The pulmonary shunt was significantly overestimated when the "gold standard" blood gas results were not used (range 0.8-9.9%). Glass (not plastic) syringes should be used in the 100% oxygen test. The syringe should be cooled immediately, even when the sample is analysed as soon as possible.     

Trophoblast IFN-tau differentially induces lymphopenia and neutropenia in lambs

Type I interferons (IFN), including IFN-alpha and IFN-beta, cause severe lymphopenia, resulting from altered lymphocyte recirculation and redistribution. IFN-tau, a product of trophectoderm of ruminant conceptuses and new member of the type I IFN family has not been examined for its effect on leukocyte recirculation. Additionally, differential effects of type I IFNs on the redistribution and recirculation of subsets of T cells have not been reported. The present study determined the effects of IFN-tau on the redistribution and recirculation of ovine leukocytes and T cell subsets. Total peripheral blood leukocytes, lymphocytes, and segmented neutrophils were reduced (p < 0.05) following treatment of lambs with IFN-tau. Furthermore, administration of IFN-tau caused an acute, differential reduction in peripheral blood CD4+ T cells (p < 0.05), CD5+ cells (p < 0.05), and gammadelta TCR+ (p < 0.01) T cells but had no effect on CD8+ T cells (p > 0.05). IFN-tau reduced the percentage of gammadelta T cells by 8-fold and that of CD4+ T cells and CD5+ cells by <2-fold in peripheral blood when compared with control lambs. The reduction in leukocytes, lymphocytes, and neutrophils was observed as early as 6-12 h after administration of IFN-tau, but levels returned to control values within 48 h. These results indicate that IFN-tau, like other members of the type I IFN family, can have immediate effects on leukocyte recirculation and redistribution. The present study is the first to demonstrate that IFN-tau differentially regulates T cell recirculation with the greatest effect on gammadelta TcR+ T cells.     

Value of measuring diurnal peak flow variability in the recognition of asthma: a study in general practice

DnaJ is a molecular chaperone, which contains a zinc finger-like motif and cooperates with DnaK to mediate the folding of newly synthesized and denatured proteins. DnaJ was overproduced and purified using the maltose binding protein (MBP) fusion vector. The fusion protein (MBP-DnaJ) was expressed in a soluble form in Escherichia coli and purified to homogeneity using amylose resin in a single step. The UV-visible absorption spectrum of MBP-DnaJ showed peaks at 355 and 475 nm. Moreover, these absorption peaks disappeared upon treatment with ethylenediaminetetraacetic acid (EDTA) or p-hydroxymercuriphenylsulfonic acid (PMPS). Inductively coupled plasma (ICP) spectrometry demonstrated that MBP-DnaJ contains Fe ions as well as Zn ions. MBP-DnaJ mediated the replication of the lambda phage in vivo, stimulated the ATPase activity of DnaK and prevented the aggregation of denatured rhodanase, indicating that fusion of MBP to the N-terminal of DnaJ does not affect the functions of DnaJ. To study the roles of bound metal ions, metal-free MBP-DnaJ, and MBP-DnaJ containing 2 Zn ions were prepared. MBP-DnaJ containing Fe and Zn ions, and MBP-DnaJ containing 2 Zn ions stimulated the ATPase activity of DnaK, prevented the aggregation of denatured rhodanase and bound to DNA to similar extents. On the other hand, metal-free MBP-DnaJ showed much lower DNA-binding ability and lower ability to prevent rhodanese aggregation. Therefore, the bound metal species do not affect the function of the zinc finger-like motif of DnaJ, whereas removal of the metal ions from DnaJ diminishes its binding ability as to DNA and denatured proteins.     

The force-frequency relationship is altered in regenerating and senescent rat skeletal muscle

Maximal tetanic tension was elicited at 200, 150, and 150 Hz in control tibialis anterior muscles and at 150, 100, and 100 Hz in 14-day regenerating muscles of young (3 months), adult (18 months), and old (31 months) Fischer 344/Brown Norway F1 rats, respectively. In contrast to young rats, increasing stimulation frequency from 50 to 150 Hz did not elicit significantly greater tetanic tension in control or regenerating muscles of old rats. At higher stimulation frequencies, tetanic fade was prevalent in control and regenerating muscles of adult (250-300 Hz) and old rats (200-300 Hz), but was only present at 14 days of recovery in regenerating muscles of young rats (300 Hz). The decreased efficacy of rehabilitative and physical medicine procedures in adult and elderly patients who have suffered skeletal muscle injury could be explained, in part, by the postulate that tetanic fade is indicative of inadequate synaptic transmission.     

Plasma lipoprotein distribution of liposomal nystatin is influenced by protein content of high-density lipoproteins

The plasma lipoprotein distribution of free nystatin (Nys) and liposomal nystatin (L-Nys) in human plasma samples with various lipoprotein lipid and protein concentrations and compositions was investigated. To assess the lipoprotein distributions of Nys and L-Nys, human plasma was incubated with Nys and L-Nys (equivalent to 20 microg/ml) for 5 min at 37 degreesC. The plasma was subsequently partitioned into its lipoprotein and lipoprotein-deficient plasma fractions by step-gradient ultracentrifugation, and each fraction was analyzed for Nys content by high-pressure liquid chromatography. The lipid and protein contents and compositions of each fraction were determined with enzymatic kits. Following the incubation of Nys and L-Nys in human plasma the majority of Nys recovered within the lipoprotein fractions was recovered from the high-density lipoprotein (HDL) fraction. Incorporation of Nys into liposomes consisting of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol significantly increased the percentage of drug recovered within the HDL fraction. Furthermore, it was observed that as the amount of HDL protein decreased the amounts of Nys and L-Nys recovered within this fraction decreased. These findings suggest that the preferential distribution of Nys and L-Nys into plasma HDL may be a function of the HDL protein concentration.     

Dynamic Modeling of Bentazon Removal by Pseudo-Moving-Bed Granular Activated Carbon Filtration Applied to Full-Scale Water Treatment

A model for the removal of pesticides by granular activated carbon (GAC) filtration in full-scale water treatment is presented. The model describes GAC filtration in a pseudo-moving-bed configuration, where two filters are operated in series and after breakthrough the first filter is regenerated and becomes the second filter. The influent of the second filter is changing due to gradual breakthrough of the first filter. Therefore, a dynamic model is developed based on kinetics, equilibrium, and mass balance equations. The model is calibrated and validated on data of full-scale and pilot plants. Operational strategies are evaluated of two different cases. From this study it can be concluded that a dynamic mathematical model can be successfully used to evaluate the performance and operation of full-scale GAC filters for pesticide removal and can be used for operational decision support. Data obtained from practice can be used for calibration without additional laboratory work.     

Synthesis and biochemical characterization of an analogue of CheY-phosphate, a signal transduction protein in bacterial chemotaxis

CheY is a signal transduction protein of the bacterial chemotaxis system that acts as a molecular switch to alter the swimming behavior of the bacterium. When CheY becomes phosphorylated at Asp57, CheY-Pi interacts with flagellar motor proteins, including FliM, to increase the likelihood that the flagellar motor will change its sense of rotation, increasing the frequency of tumbling. The structure of CheY in its dephosphorylated (inactive) state has been intensively investigated. The short lifetime ( approximately 20 s) of the aspartyl phosphate has precluded the complete structural determination of CheY-Pi. We have synthesized an analogue of CheY-Pi by alkylating an aspartate-to-cysteine mutant at position 57 of CheY to add a phosphonomethyl group at Cys57. This analogue, phosphono-CheY, is stable for months. Phosphono-CheY binds to two of the targets of CheY-Pi, FliM and CheZ, in a manner similar to that of CheY-Pi and much better than either unphosphorylated CheY or the unmodified form of D57C CheY. Phosphono-CheY also binds Mg(II) with a dissociation constant of approximately 6 mM at neutral pH and moderate salt level. These observations indicate that phosphono-CheY is a good biochemical analogue of CheY-Pi.     

Structural characterization of four genetic variants of human serum albumin associated with alloalbuminemia in Italy

A long-term electrophoretic survey on plasma proteins, which was carried out in several clinical laboratories in Italy, identified 28 different genetic variants of human serum albumin and four cases of analbuminemia. We have previously characterized 16 point mutations, 3 C-terminal mutants, and the genetic defects in two analbuminemic subjects. Here, we report the molecular defects of four alloalbumins that have been characterized by protein structural analysis. Of these, three represent new single-point mutations: albumins Tregasio, Val122-->Glu, Bergamo, Asp314-->Gly, and Maddaloni, Val533-->Met. The fourth, albumin Besana Brianza, has the same Asp494-->Asn mutation that introduces a glycosylation site which has been previously reported in a variant from New Zealand, albumin Casebrook. However, in contrast to albumin Casebrook, albumin Besana Brianza is only partially glycosylated and the oligosaccharide is heterogeneous, consisting of a biantennary complex type N-glycan with either two or one sialic acid residue(s) on the antennae. Both albumin Maddaloni and Besana Brianza represent mutations at hypermutable CpG dinucleotide sites; albumin Maddaloni is a mutant that does not involve a charged amino acid.