Neutrophil alkaline phosphatase (NAP) was analysed in 25 pregnant women with trisomy 21 foetuses whose chromosomal aberration was recognized by cytogenetic study after amniocentesis. Enzyme investigation was performed at 20-22 weeks of gestation using cytochemical and biochemical techniques. Twenty-nine women at the same stage of normal pregnancies were selected as controls. In parallel, each mother was karyotyped. Ten subjects from each series underwent biochemical and immunological investigation: measurement of enzyme levels, thermostability study and immunological tests with alkaline phosphatase isoenzyme antibodies. NAP from pregnant women with trisomy 21 foetuses was characterized by: (1) a lower rate of enzyme activity, (2) a large amount of heat-stable enzyme (T = 56 degrees C for biochemical assays, T = 85 degrees C for cytochemical tests), and (3) a marked loss of liver antigenicity. These findings suggest the presence in trisomy 21 pregnancies of a non-specific alkaline phosphatase isoenzyme which appears as an "enzyme marker" in maternal circulating neutrophils.

Alkaline phosphatase concentrations are known to increase in blood neutrophils of normal pregnant women. The main kinetic parameters of this enzyme were analysed and compared in a group of 30 women with normal pregnancies and a group of 11 women whose fetuses had trisomy 21 (Down's syndrome = DS). The subjects were studied at an identical stage of gestation. Significant changes occurred in thermal stability and urea resistance in cases of DS pregnancies. We also investigated the inactivation constants for two chemicals: L-p-bromotetramisole, an uncompetitive inhibitor, and sodium thiophosphate, a competitive inhibitor. Ki measured for the two inhibitors were found to be significantly lower in cases of pathological pregnancies. The patterns observed in inhibition constants extend the biochemical characteristics of the atypical isoenzyme expressed in neutrophils of women with DS pregnancies.

Biochemical, cytochemical characteristics and electron microscopy subcellular distribution of neutrophil alkaline phosphatase (NAP) were analysed in blood and/or smear samples from 39 trisomy 21 patients (Down's syndrome) aged 11.5-18 years (mean 15.5 years) and 55 normal subjects aged 12-20.5 years (mean 17 years). All patients were karyotyped. NAP cytochemical procedures were carried out on all subjects; biochemical NAP determinations were made in 10 patients and 20 controls; ultrastructural electron microscopy of AP was performed in three patients and four normal subjects. Neutrophil alkaline phosphatase from patients with trisomy 21 displayed the following changes: (1) a significant increase of enzyme activity, (2) a high thermal lability of enzyme. Electron microscope morphology exhibited large deposits of NAP reaction product associated with the plasma membrane and intracellular main organelles, like phosphasomes. The NAP biochemical and cytochemical characteristics suggest that trisomy 21 neutrophils contain a non-specific AP isoenzyme, closely related to the early placental form.

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Background Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8 th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein. Results The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature. It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent. Conclusion It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.

Assays of neutrophil phosphotyrosine phosphatase activity and determination of haematological parameters were performed on 12 trisomic 21 probands without any clinical or biological symptom of other evolutive disease. Haematological studies showed the two main classical abnormalities: the existence of a macrocytosis and an enhanced lymphocyte count. Of interest are the very reduced rates of phosphotyrosine phosphatase activity found in granulocytes from these patients. This defect in protein phosphatase can be considered as an additional enzymatic change extending the list of modified factors recognized at molecular and cellular levels in subjects whose risk of leukaemia is significantly increased.

The study was performed on dog thyroid slices after in vivo administration of monocomponent insulin (carotid perfusion of a lobe: 25 mIU/kg/min for 15 min, and 2nd lobe perfused as control: saline) and after in vitro hormone addition (100 mIU/ml of glucose-free incubation medium). The 3H-leucine incorporation in thyroid proteins (dpm/mg) was determined following incubation of slices for 1 h and 2 h. Insulin given in situ significantly increased the mean incorporation into homogenate and cytosol proteins: about 130% after 1 h of incubation, for an identical hypoglycaemia in both lobes. When insulin was added to the medium, a similar response was observed after 1 h of incubation, the effect being less pronounced after 2 h but still significant. The sucrose gradient analysis of the soluble fraction showed that the stimulation affected all thyroid proteins. Polyribosomes isolated from in vivo insulin-perfused thyroids exhibited an increased activity when tested in cell-free system. These data suggest that insulin stimulated the thyroid protein synthesis in dogs through a direct action on the thyroid cell.

Background: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8 th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein.