Sandwich-type deoxyribonucleic acid hybridization assays based on enzyme amplified time-resolved fluorometry

We report microtiter well-based sandwich-type DNA hybridization assays using enzyme amplified time-resolved fluorometry of Tb3+ chelates. The target DNA was hybridized with two adjacent and non-overlapping oligonucleotide probes, one oligonucleotide serving as the capture probe and the other as the detection probe. Two ligand-specific binding protein pairs were used alternately for capture of the hybrids to the solid phase and detection; the biotin-streptavidin and the digoxigenin-anti-digoxigenin interaction. In both cases, alkaline phosphatase was used as a reporter molecule and diflunisal phosphate as a substrate. The catalytic hydrolysis of the substrate produces diflunisal which forms ternary fluorescent complex with Tb(3+)-EDTA. Furthermore, we studied the effect of the probe labeling method and the position of the label on the sensitivity of the assays. The data suggest that capture of the hybrids through biotin-streptavidin and detection via digoxigenin-anti-digoxigenin offer 2-3 times higher sensitivity than the reverse configuration. The highest sensitivity was achieved with enzymatic labeling of capture and detection probes at the 3' end. A signal-to-background ratio of 4 was achieved for 0.2 fmol of target DNA. The RSD were better than 4%.     

Cerebral apoplexy in children

Stroke in children is rare. No really good studies of the incidence are available, an estimate, however, is 2-3/100,000 children per year. In this paper we discuss the pathophysiology and the many different causes of stroke in children. In many of the cases more than one prothrombotic condition exists. If the cause is not obvious an extensive programme of examinations is recommended. This is important not only in order to give the best individual treatment, but also necessary in order to decide whether the stroke has a genetic cause. Initially, the treatment is symptomatic, attaching importance to achieving good perfusion of the cerebrum and lowering the energy consumption of the cerebrum. The rational treatment might be prompt reperfusion by thrombolytic medicine: this regime has been tried in adults, but as yet no consensus about this treatment modality exists. In some cases causal treatment is possible. If the stroke has a genetic cause genetic consultation is important and prenatal investigations might be possible. Overall, the mortality in stroke in children is about 25%. About 25% will live without any sequelae and approximately 50% of the children will disabled to a greater or lesser extent.     

Effects of a new platelet glycoprotein IIb/IIIa antagonist, SR121566, on platelet activation, platelet-leukocyte interaction and thrombin generation

The effects of SR121566, a new inhibitor of the glycoprotein (GP) IIb/IIIa complex on platelet activation and platelet-leukocyte interactions, as well as on thrombin generation were investigated. SR121566 dose-dependently inhibited adenosine diphosphate (ADP)-induced platelet fibrinogen binding determined either by flow cytometry analysis (IC50=50 nmol/l) or by measuring the binding of 125I-fibrinogen to activated human gel-filtered platelets (IC50=20 nmol/l). Consistent with its inhibitory effects on platelet fibrinogen binding, SR121566 demonstrated a dose-dependent inhibition of collagen-, ADP- or thrombin-induced platelet aggregation with IC50 values ranging between 20 and 60 nmol/l. SR121566, even tested at high concentrations, did not significantly affect ADP-induced platelet-leukocyte aggregate formation. The GPIIb/IIIa antagonist strongly inhibited thrombin generation in both native clotting blood and recalcified whole blood, suggesting that SR121566, by interfering with the platelet-activation events involved in facilitating thrombin generation, may also function as an anticoagulant, an effect which may contribute to its antithrombotic properties in humans.     

Selective inhibition of cyclic AMP-dependent protein kinase by isoquinoline derivatives

A large series of isoquinoline derivatives was synthesised including derivatives of isoquinoline, isoquinolino[3,4-c]furazan, 1,2-dihydro-1-oxoisoquinoline, 6-oxopyrimido[1,2-d]isoquinoline, benzo[c][1,8]-naphthyridine, pyrazino[2,3-c]isoquinoline and benzimidazo[2,1-a]isoquinoline as well as further structurally related isoquinoline derivatives and pyrido-2,3-furazans. Representatives of all of these classes of isoquinolines are potent and selective inhibitors of the cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK) from rat liver. The most effective cAK inhibitors are a series of 1,3-di-substituted and 1,3,4-tri-substituted isoquinolines (IC50 values 30-50 nM) (compounds A1, A2, A3, A4 and A5) and 2-ethylcarboxy-3-amino-5,6-dihydro-6-oxobenzo[c] [1,8]naphthyridine (E1) (IC50 0.08 microM). Compounds A1-A5 inhibit cAK in a fashion that is competitive with respect to ATP as substrate. The isoquinoline inhibitors A1-A5 are ineffective or very poor inhibitors of wheat embryo Ca(2+)-dependent protein kinase (CDPK) and rat brain Ca(2+)-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK) and potato tuber cyclic nucleotide-binding phosphatase (Pase). E1 is a moderately effective inhibitor of CDPK and PKC (IC50 values 30 and 61 microM, respectively). The bisisoquinoline-1(2H)-one compound B7 inhibits cAK, CDPK, PKC and MLCK (IC50 values 8, 95, 24 and 7 microM, respectively) as does J1 [2-(p-bromophenyl)pyrrolo-[2,3-c]isoquinoline-5(4H)-one] (IC50 values 2, 50, 44 and 7 microM, respectively). The very potent isoquinoline-derived cAK inhibitors found here involve substitution of the N-containing isoquinoline ring system and these inhibitors show high specificity for cAK.     

At-line solid-phase extraction for capillary electrophoresis: application to negatively charged solutes

The molecular karyotype of a series of Giardia lamblia isolates representing the two major genotypes (Groups 1 and 3) was generated by assigning 13 genetic markers to chromosomes separated by pulsed-field gel electrophoresis. The co-localization identified five linked groups of genetic markers in Group 1 isolates. For each of the five linkage groups, there were up to four size variants that hybridized with the same genetic markers. Long range physical maps of the regions flanking the low copy number genetic markers indicated that these size variants were homologous chromosomes. The linkage groups were similar in Group 1 and 3 isolates. The core of each chromosome was stable while the subtelomeres were variable. The location of the ribosomal DNA repeats was variable among the different isolates and they were found in the subtelomeric regions of any of the five linkage groups. The data suggest a functional ploidy of at least four. Hypervariable subtelomeric regions of homologous chromosomes provide the structural basis of the chromosome size heterogeneity that is characteristic of G. lamblia.     

Parasolitary nucleus: a source of GABAergic vestibular information to the inferior olive of rat and rabbit

At least two subnuclei of the inferior olive, the beta-nucleus, and the dorsomedial cell column (dmcc), contain vestibularly responsive neurons that receive a dense descending projection that uses gamma-aminobutyric acid (GABA) as the transmitter. In contrast to the GABAergic innervation of other olivary subnuclei, the terminal boutons that terminate on neurons in the beta-nucleus and the dorsomedial cell column remain intact after cerebellectomy, ruling out both the cerebellum and the cerebellar nuclei as afferent sources. By using both immunohistochemical as well as orthograde and retrograde tracer methods, we have identified the source of the GABAergic pathway to the beta-nucleus and dmcc in both rat and rabbit. Under physiologic recording of single olivary neurons to guide electrode placement, we injected the bidirectional tracer, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the beta-nucleus and dmcc of the inferior olive. These injections retrogradely labeled neurons in the parasolitary nucleus (Psol) near the vestibular complex. Psol neurons were identified as GABAergic with an antibody to glutamic acid decarboxylase (GAD). In the rat, Psol neurons are small (5-7 microm in diameter) and number approximately 1,800. In the rabbit, they are slightly larger (6-9 microm in diameter) and number approximately 2,200. WGA-HRP injections in conjunction with GAD immunohistochemistry double labeled a high percentage of neurons in both the rat and rabbit Psol. Injection of the orthograde tracer Phaseolus vulgaris-leucoagglutinin into the area of the Psol revealed a projection from this region to both the beta-nucleus and dmcc. Subtotal electrolytic lesions of this division of the Psol caused a substantial reduction in GAD-positive synaptic terminals in both the ipsilateral beta-nucleus and dmcc. The location of these GABAergic neurons, bordering both the nucleus solitarius and caudal vestibular complex, emphasizes the importance of the Psol in the processing of both vestibular and autonomic information pertinent to postural control.     

Working together in Peru--language needn''t be a problem!

The antihypoxic, anti-ischemic and antianginal activity of biotechnological cytochrome c which had been immobilized on a dialdehydedextran and polyethyleneglycol carriers was studied in various experimental animals (mice, rats, rabbits). The immobilized cytochrome c exhibits higher antihypoxic and anti-ischemic activity on different models compared to cytochrome c.     

Microglial and macrophage reactions mark progressive changes and define the penumbra in the rat neocortex and striatum after transient middle cerebral artery occlusion

Transient middle cerebral artery occlusion in rats leads to infarction of the lateral part of the striatum and adjacent neocortex, with selective neuronal necrosis in the bordering penumbral zones. Administration of glutamate, cytokine, and leukocyte antagonists have rescued mainly neocortical neurons, indicating differences in the degenerative processes. The aim of this study was, therefore, to describe the microglial/macrophage activation and polymorphonuclear leukocyte recruitment patterns and to correlate these with the ischemia-induced degenerative processes. The analysis showed significant differences in the characteristics and timing of the microglial/macrophage responses between the caudate putamen and neocortical infarct zones, the infarct zones and their associated penumbral zones, as well as between the striatal and the neocortical penumbral zone. Infiltrations with polymorphonuclear leukocytes into the infarct zones were limited and shortlasting and confined to the acutely degenerating striatum and piriform cortex. A delayed, massive infiltration with lipid phagocytes into the caudate putamen infarct markedly contrasted an early recruitment and activation of microglia/macrophages in the adjacent penumbra. Within the neocortex, a later onset of degeneration along the insular-parietal axis was marked by neuronal expression of heat shock protein and a progressive microglial activation with induction of the full repertoire of microglial activation markers, including a widespread microglial major histocompatibility complex (MHC) class II antigen expression. We interpret the present results as delineating two differentially progressing penumbral zones, which are likely to reflect differences in the underlying degenerative processes. Differences in the microglial/macrophage activation pattern attract special attention, as these cells may constitute specific targets for therapeutic intervention.