Stefano Biagioni

Membrane acetylcholinesterase in murine muscular dystrophy In vivo and in cultured myotubes

Murine muscular dystrophy is characterized by a reduction of the 10S molecular form of acetylcholinesterase (AChE); this reduction occurs in both strains of dystrophic mice and at the time of the phenotypic appearance of the disease.

Alpha 1-antitrypsin, transferrin, alkaline phosphatase, phosphohexoseisomerase and gamma-glutamyltransferase in breast cyst fluid.

The levels of α1-antitrypsin, transferrin, alkaline phosphatase, phosphohexoseisomerase and γ-glutamyltransferase were measured in 32 samples of breast cyst fluid, and a wide range of values was obtained. The levels observed in some samples for these parameters, being similar to those of normal serum, might suggest a mechanism of plasma exudation for the formation of breast cyst fluid. Nevertheless, a comparison with the maximum normal serum reference value revealed very high levels of γ-glutamyltransferase in all of the samples; about 50% also exhibited high levels of phosphohexoseisomerase. These results indicate that the formation of breast cyst fluid takes place with a specific local production, in addition to the mechanism of selective plasma exudation. Moreover, elevated transferrin levels in some cyst fluids are probably the expression of a high biosynthetic activity and could be diagnostically useful in the course of breast gross cystic disease. The importance of these observations from the point of view of diagnostic and prognostic trends are discussed.

5-Hydroxytryptamine 1A and 2B Serotonin Receptors in Neurite Outgrowth: Involvement of Early Growth Response Protein 1

Neurotransmitters play important roles in neurogenesis; in particular, acetylcholine and serotonin may regulate neurite elongation. Acetylcholine may also activate transcription factors such as early growth response protein 1 (EGR-1), which plays a role in neurite extension. N18TG2 neuroblastoma cells (which do not produce neurotransmitters and constitutively express muscarinic acetylcholine receptors) were transfected with constructs containing the cDNA for choline acetyltransferase, 5-hydroxytryptamine 1A (5-HT1A) and 5-HT2B serotonin receptors to study acetylcholine and serotonin interplay in neurite outgrowth. 5-HT1A receptor stimulation causes a decrease in EGR-1 levels and inhibition of neurite outgrowth; 5-HT2B stimulation, however, has no effect. Muscarinic cholinergic stimulation, on the other end, increases EGR-1 levels and fiber outgrowth. Inhibition of EGR-1 binding reduces fiber outgrowth activity. When both cholinergic and 5-HT1A receptors are stimulated, fiber outgrowth is restored; therefore, acetylcholine counterbalances the inhibitory effect of serotonin on neurite outgrowth. These results suggest that EGR-1 plays a role in the interplay of acetylcholine and serotonin in the regulation of neurite extension during development.

Muscle acetylcholinesterase in childhood myopathies.

The activities and the molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were examined in 28 biopsies of quadriceps femoris muscle from children with a myopathic non-dystrophic disease. These cases were compared with biopsies from 7 children with a neurogenic damage, 14 children with muscular dystrophy and 12 controls. All the biopsies, histochemically stained for AChE, showed no endplates; electron microscopy of muscle fibers from diseased biopsies revealed a diffuse AChE reaction on the fiber surfaces which was not associated with any endplate structure. The AChE activities in NaCl/Triton X-100 extracts from the three groups of patients were all more or less the same, and average levels were similar to those evidenced in controls. The complete disappearance of heavy and medium forms of AChE was noted in 60% of myopathic non-dystrophic patients. We never observed the pattern characteristic to these patients in the biopsies from neurogenic and dystrophic patients or from controls, which displayed a high variability in the profiles of AChE molecular forms.

Alkaline Phosphatase Expression in Human Chorionic Villi

The physicochemical properties and electrophoretic mobility of different isoforms of alkaline phosphatase were studied in chorionic villi. Based on selective inactivation and inhibition studies (thermal stability, inactivation by urea, EDTA and L(+)ascorbic acid and L-amino acid inhibition), evidence was obtained for the existence of two distinct types of alkaline phosphatase in trophoblast cells. One type is peculiar to chorionic villi while the other is also found in term placenta. Both show two isoforms. These two isoforms were observed with polyacrylamide gel electrophoresis, carried out at pH 6.0 and 9.5. It is suggested that the qualitative and quantitative methods of alkaline phosphatase analysis could be used for first trimester fetal diagnosis of severe infantile hypophosphatasia and for understanding genetic control during early fetal development.

Muscle Acetylcholinesterase in a Familial Myopathic Disease

Three sisters with myopathy characterized by different degrees of weakness, hypotonia, cramps and a significant hypertrophy of the calves underwent clinical tests. Laboratory examinations (nerve conduction velocity, electromyography and serum enzymes), serial histochemical analyses of muscle specimens and tests for muscular acetylcholinesterase (AChE) activity and its molecular forms were performed. AChE activities did not differ significantly from those of controls, while sedimentation patterns evidenced the disappearance of 16 S, 13 S and 10 S molecular forms in the elder sisters. The genealogical tree of the patients is described and their cases compared to those of others with calf hypertrophy reported in the literature.

Cholinesterase evaluation in the prenatal diagnosis of open neural tube defects

Summaryα-fetoprotein (AFP) and cholinesterase levels in amniotic fluid were determined and the efficiency of these laboratory tests in the prenatal diagnosis of neural tube defects was examined. Using the AFP test with cut-off levels correlated to gestational age, we have detected 8 cases of neural tube defects and one case of abdominal wall defect in about 1,200 pregnancies; false-negative values were absent. Acetylcholinesterase (AChE) and butyrylcholinesterase activities were measured and the electrophoretic pattern of AChE was examined in 100 amniotic fluid samples. The diagnosis of neural tube defects was always confirmed. There were no diagnostic problems due to blood-contaminated amniotic fluid samples. The results obtained using different quantitative methods for the determination of cholinesterase activity, as well as the potential use of these tests in routine examinations, are discussed.

Hydroxyproline and creatinine levels in normal amniotic fluid

SummaryTotal hydroxyproline and creatinine concentrations as well as their ratios were determined in 29 amniotic fluid samples from normal pregnancies between the 16th and 20th week of gestation. Total hydroxyproline and creatinine levels, and their ratios, were not statistically different considering either the values at each week or those of the whole 5-week period. Our results, compared with the few others reported in the literature, can provide indications useful for defining the normal range in amniotic fluid, in relation to the weeks of gestation.

Egr-1 Maintains NSC Proliferation and Its Overexpression Counteracts Cell Cycle Exit Triggered by the Withdrawal of Epidermal Growth Factor

In adult mammals, neural stem cells (NSCs) reside in specialized niches at the level of selected CNS regions, such as the subventricular zone (SVZ). The signaling pathways that regulate NSC proliferation and differentiation remain poorly understood. Early growth response protein 1 (Egr-1) is an important transcription factor, widely studied in the adult mammalian brain, mediating the activation of target genes by a variety of extracellular stimuli. In our study, we aimed at testing how Egr-1 regulates adult NSCs derived from mouse SVZ and, in particular, the interplay between Egr-1 and the proliferative factor EGF. We demonstrate that Egr-1 expression in NSCs is induced by growth factor stimulation, and its level decreases after EGF deprivation or by using AG1478, an inhibitor of the EGF/EGFR signaling pathway. We also show that Egr-1 overexpression rescues the cell proliferation decrease observed either after EGF removal or upon treatment with AG1478, suggesting that Egr-1 works downstream of the EGF pathway. To better understand this mechanism, we investigated targets downstream of both the EGF pathway and Egr-1, and found that they regulate genes involved in NSC proliferation, such as cell cycle regulators, cyclins, and cyclin-dependent kinase inhibitors.