Comparison of the carbohydrate and amino acid composition of normal and S-variant alpha-1-antitrypsin.

alpha-1-Antitrypsin has been isolated and purified from the serum of an individual with the Pi S phenotype whose serum contains only 50--60% as much alpha-1-antitrypsin as normal M-type serum. The preparation was homogeneous by the criteria of sodium dodecyl sulfate polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrigufation. When analyzed in the ultracentrifuge, the S-type alpha-1-antitrypsin exhibited a molecular weight of 47,500 which was essentially the same as that of the M-type (47,300) and the Z-type (47,500) alpha-1-antitrypsin. The S-type alpha-1-antitrypsin contains 15.2% carbohydrate consisting of 16.4 residues/mol of N-acetylglucosamine, 7.8 residues/mol of mannose. 6.7 residues/mol of galactose and 7.1 residues/mol of sialic acid which is essentially the same as the carbohydrate composition of the M-type alpha-1-antitrypsin. In addition, M- and S-type alpha-1-antitrypsin have very similar amino acid compositions.     

Multiregional evaluation of the SimPlate heterotrophic plate count method compared to the standard plate count agar pour plate method in water

A new SimPlate heterotrophic plate count (HPC) method (IDEXX Laboratories, Westbrook, Maine) was compared with the pour plate method at 35 degrees C for 48 h. Six laboratories tested a total of 632 water samples. The SimPlate HPC method was found to be equivalent to the pour plate method by regression analysis (r = 0. 95; y = 0.99X + 0.06).     

Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathione-orthophthalaldehyde adduct

Glutathione reacts with orthophthalaldehyde to form a stable, highly fluorescent tricyclic derivative which is easily separated and quantitated by high-performance liquid chromatography. Separation of the glutathione adduct is achieved by isocratic elution over a reverse-phase column with 7.5% methanol/92.5% 0.15 M sodium acetate, pH 7.00. The adduct is detected fluorometrically and quantitated by integration of peak area. Detection of 0.1 to 200 pmol glutathione produces a linear response and the recovery of reduced and oxidized glutathione from rat liver homogenate, bile, and plasma is quantitative. The chemical identity of the adduct was confirmed by mass spectrometry.     

Purification and partial characterization of a 19kD/pI 4.5 nucleolar phosphoprotein

Two-dimensional PAGE analysis of proteins associated with the slowly sedimenting fibrillar structures of HeLa nucleoli revealed a protein with a M of 19,000 and a pI of 4.5 which was highly labeled both with 32P-orthophosphate and 35S-methionine. The protein was isolated from Novikoff hepatoma nucleoli by extraction in 0.35 M NaCl and 5 mM DTT followed by chromatography in EDTA on DEAE-cellulose and Sephadex G-100. The protein was homogeneous with respect to two-dimensional PAGE, number of tryptic peptides and carboxyl terminal analysis. The protein contained an acidic/basic amino acid ratio of 2.1, 7 residues of methionine, 2 residues of cysteine, a blocked amino terminus and a carboxyl terminal lysylleucine.     

Mutations in the gene PRRT2 cause paroxysmal kinesigenic dyskinesia with infantile convulsions

Paroxysmal Kinesigenic Dyskinesia with Infantile Convulsions (PKD/IC) is an episodic movement disorder with autosomal dominant inheritance and high penetrance, but the causative gene is unknown. We have now identified four truncating mutations involving the PRRT2 gene in the vast majority (24/25) of well characterized families with PKD/IC. PRRT2 truncating mutations were also detected in 28 of 78 additional families. The PRRT2 gene encodes a proline-rich transmembrane protein of unknown function that has been reported to interact with the t-SNARE, SNAP25. PRRT2 localizes to axons but not to dendritic processes in primary neuronal culture and mutants associated with PKD/IC lead to dramatically reduced PRRT2 protein levels leading ultimately to neuronal hyperexcitability that manifests in vivo as PKD/IC.