Charles-Michel Wolff

Stabilization of dry immobilized acetylcholinesterase on nitrocellulose membrane for rapid colorimetric screening of its inhibitors in water and biological fluids

Abstract We show the ability of a gelatin and a BSA-trehalose film to convert normally fragile dry immobilized acetylcholinesterase enzyme (AchE) into a stable reagent on nitrocellulose membrane. The remarkable property of the dry immobilized AchE enzyme preparation is its stability when exposed to temperatures as high as 50°C. The proposed method offers a rapid, simple, and inexpensive means in a non-laboratory setup for qualitative analytical colorimetric screening of AchE inhibitor residues such as pesticides and drugs in water and biological fluids.

Significance of dinucleoside tetraphosphate production by cultured tumor cells exposed to the presence of ethanol.

The use of 30 to 50% ethanol solutions to extract the nucleotides from HTC and A-459 cells results in dinucleoside tetraphosphate (Ap4X) levels 3-30-fold as high as those obtained by 5% classical trichloracetic acid extraction, while ATP levels are identical in both cases. The amplification factor varies with the percentage of ethanol and duration of contact between the cells and the extraction mixture. It remains constant for the HTC cells during cell growth, but exhibits a maximum for the A-459 cells towards the end of the exponential growth period. The incorporation of radioactivity in Ap4X when [alpha-32P]ATP is added to the extraction mixture suggests an Ap4X neosynthesis in the presence of ethanol. The results carried out in the presence of pyrophosphate, EDTA and zinc acetate strongly suggest that aminoacyl-tRNA synthetases could be responsible for the increase in Ap4A content with ethanol treatment. Nevertheless, the effect of ethanol is probably not the result of an activation of these enzymes, but rather, as already suggested by earlier results in our laboratory, the result of a fast inactivation of the degradation enzymes.

Protection of immunoreactivity of dry immobilized proteins on microtitration plates in ELISA: application for detection of autoantibodies in myasthenia gravis.

We show the ability of the BSA-trehalose film to convert normally fragile proteins such as mouse monoclonal antibody to the Alzheimer precursor protein A4 (APP695) and cell line TE671 acetylcholine receptor (AChRTE671) into a stable reagent, after its immobilization on microtitration plates. The remarkable property of the dry immobilized proteins are their stability under prolonged exposure to temperatures as high as 50 degrees C. Using the AChRTE671, the proposed method was applied for the measurement of anti-AChR autoantibodies in Myasthenia gravis by means of an enzyme-linked immunosorbent assay (ELISA). The test was shown to be specific and able to detect anti-AChR autoantibodies at concentrations as low as 3 nM. Using the same AchRTE671 as antigen, the results of examination of 34 serum samples for detection of anti-AChR autoantibodies by ELISA were compared with those of the conventional radioimmunoprecipitation assay (RIA). It was concluded that ELISA is another useful method for the diagnosis of M. gravis. The ELISA method offers a rapid, simple, safe and inexpensive means for mass screening of M. gravis.

Cloning and Expression of the Surfeit Locus Member Surf-6 During Embryogenesis in Xenopus laevis

The Surf-6 gene, already characterized in fish ( Fugu rubripes ), mouse and man, is a member of the surfeit locus and encodes a nucleolar-matrix protein, which is ubiquitously expressed. This gene has been isolated in Xenopus laevis and shows high sequence similarity to its orthologues in other species. The putative protein is 342 amino acids long and several motifs are conserved, particularly a potential nuclear localization signal. During embryogenesis, after an initial decrease in the expression of the maternal Surf-6 RNA, the level increases to reach a maximum at hatching. The global level of the Surf-6 transcript at early neurula is enhanced by the overexpression of fli, a member of the ets gene family.

UTILIZATION OF NUCLEOTIDE PROBES FOR THE MEASUREMENT OF SPECIFIC MESSENGER RNA: APPLICATION FOR MOLECULAR DIAGNOSIS OF AUTOSOMAL RECESSIVE SPINAL MUSCULAR ATROPHY

ABSTRACT Spinal muscular atrophy (SMA) is a lethal autosomal recessive disease. SMA is characterized by degeneration of motor neurons in the spinal cord, causing progressive weakness of the limbs and trunk, followed by muscle atrophy. The gene most highly associated with SMA is the survival motor neuron (SMN) gene. This paper describes the results concerning the development of a quantitative method for the molecular diagnosis of SMA by measuring the amount of cytosolic mRNA from human muscle cells. The procedures using radioactive material and the Enzyme-Linked Immunosorbent Assay (ELISA) non-radioactive method were developed using 32P-dCTP labeled and biotinylated nucleotide probes, respectively; the results obtained demonstrate that the measurement of specific mRNA could be used as a quantitative method for the molecular diagnosis of SMA. There was a perfect concordance of the results obtained between the procedure using radioactive material, the ELISA method and the single-strand conformation polymorphism (SSCP) analysis regarding negative and positive SMA samples. All values obtained for the control group were significantly greater than the ones obtained for the SMA positive samples (33–76% in radioactive method and 38–54% in ELISA method). The methods developed in this study may be applicable to the diagnosis (detection of homozygous and heterozygous deletions in exons 7 and 8 of the SMN gene) and the control of mRNA concentrations in future gene therapy of patients with SMA.