Quantitative and qualitative assay of amniotic-fluid acetylcholinesterase in the prenatal diagnosis of neural tube defects

Summary In 110 amniotic fluids the specific acetylcholinesterase was determined quantitatively and qualitatively. In the quantitative assay there were a considerable number of false positives and false negatives, although the mean value of the normal controls differed significantly from that of neural tube defect pregnancies. By the electrophoretic separation of acetylcholinesterase, however, all fluid samples with borderline alpha-fetoprotein levels or fetal blood contamination could be correctly classified. With the exception of one skin-covered spina bifida all neural tube defects in the second trimester could be identified by this method. The second fast-moving band characteristic of the specific acetylcholinesterase was also present in abdominal wall defects and intrauterine death.     

Production and properties of xylanases from thermophilic actinomycetes

30 strains of xylanolytic thermophilic actinomycetes were isolated from composted grass and cattle manure and identified as members of the generaThermomonospora, Saccharomonospora, Microbispora, Streptomyces andActinomadura. Screening of these strains for extracellular xylanase indicated that strains ofSaccharomonospora andMicrobispora generally were poor xylanase producers (0.5–1.5 U/ml) whereas relatively high activities were observed in cultures ofStreptomyces andActionomadura (4–12 U/ml).A preliminary characterization of the enzymes of strains of the latter genera suggested that xylanases of all the strains ofActinomadura exhibited higher thermostabilities than those ofStreptomyces. To evaluate the potential of thermophilicActinomadura for industrial applications, xylanases of three strains were studied in more detail. The highest activity levels for xylanases were observed in cultures grown on xylan and wheat bran. The optimal pH and temperature for xylanase activities ranged from 6.0 to 7.0 and 70 to 80°C. The enzymes exhibited considerable thermostability at their optimum temperature. The half-lives at 75°C were in the range from 6.5 to 17h. Hydrolysis of xylan by extracellular xylanases yielded xylobiose, xylose and arabinose as principal products. Estimated by the amount of reducing sugars liberated the degree of hydrolysis was 55 to 65%. Complete utilization of xylan is presumably achieved by -xylosidase activities which could be shown to be largely cell-associated in the 3Actinomadura strains.     

Energy-linked reactions catalyzed by the purified ATPase complex (F0F1) from Rhodospirillum rubrum chromatophores

1. The isolation of F0F1-ATPase complex from Rhodospirillum rubrum chromatophores by the use of taurodeoxycholate is described. 2. The enzyme preparation contains about 12 polypeptides; five are subunits of the F1 moiety. 3. The ATPase activity of the purified enzyme is dependent on the addition of phospholipids. 4. Km-vales for Mg2+-ATP and Ca2+-ATP are similar to the values obtained for the membrane-bound enzyme. 5. The F0F1-ATPase complex is more than 70% inhibited by oligomycin and N,N'-dicyclohexylcarbodiimide. 6. The F0F1-ATPase complex was integrated into liposomes. The reconstituted proteoliposomes catalyzed energy transduction as shown by ATP-dependent quenching of acridine dye fluorescence and ATP-32Pi exchange.     

The ATP synthetase of Escherichia coli K12: purification of the enzyme and reconstitution of energy-transducing activities.

The ATP synthetase of Escherichia coli K12 was purified by a simple procedure. The dicyclohexylcarbodiimide-sensitive ATPase activity was enriched 21-fold. The ATP synthetase preparation contained the eight polypeptides (alpha, beta, gamma, a,delta, b,espilon, c) of the enzyme and a residual contamination (4% of the total protein) as shown by dodecylsulfate/polyacrylamide electrophoresis. The polypeptide c was specifically labelled with [14C]dicyclohexylcarbodiimide. Energy-transducing activities were reconstituted from soybean phospholipids and the purified enzyme. The proteoliposomes exhibited a significantly higher ATP-32Pi exchange activity and a higher proton-translocating activity as compared to the untreated membranes.     

Localized synthesis of specific proteins during oogenesis and early embryogenesis inDrosophila melanogaster

Summary Protein synthesis in egg follicles and blastoderm embryos ofDrosophila melanogaster has been studied by means of two-dimensional gel electrophoresis. Up to 400 polypeptide spots have been resolved on autoradiographs. Stage 10 follicles (for stages see King, 1970) were labelled in vitro for 10 to 60 min with³⁵S-methionine and cut with tungsten needles into an anterior fragment containing the nurse cells and a posterior fragment containing the oocyte and follicle cells. The nurse cells were found to synthesize a complex pattern of proteins. At least two proteins were detected only in nurse cells but not in the oocyte even after a one hour labelling period. Nurse cells isolated from stages 9, 10 and 12 follicles were shown to synthesize stage specific patterns of proteins. Several proteins are synthesized in posterior fragments of stage 10 follicles but not in anterior fragments. These proteins are only found in follicle cells. No oocyte specific proteins have been detected. Striking differences between the protein patterns of anterior and posterior fragments persist until the nurse cells degenerate. In mature stage 14 follicles, labelled in vivo, no significant differences in the protein patterns of isolated anterior and posterior fragments could be detected; this may be due to technical limitations. At the blastoderm stage localized synthesis of specific proteins becomes detectable again. When blastoderm embryos, labelled in vivo, are cut with tungsten needles and the cells are isolated from anterior and posterior halves, differences become apparent. The pole cells located at the posterior pole are highly active in protein synthesis and contribute several specific proteins which are found exclusively in the posterior region of the embryo. In this study synthesis of specific proteins could only be demonstrated at those developmental stages which are characterized by the presence of different cell types within the egg chamber, while no differences were detected when stage 14 follicles were cut and anterior and posterior fragments analyzed separately. The differences in the pattern of protein synthesis by pole cells and blastoderm cells indicate that even the earliest stages of determination are reflected by marked changes at the biochemical level.     

The use of several energy-coupling reactions in characterizing mutants of Escherichia coli K12 defective in oxidative phosphorylation.

Oxidative phosphorylation, ATP-32Pi exchange, ATP-dependent quenching of acridine-dye fluorescence, ATP-dependent transhydrogenase and ATP-dependent transport of thiomethyl beta-D-galactoside are shown to be experimentally equivalent tools to study the functional state of the ATPase complex in Escherichia coli wild-type and mutant strains defective in oxidative phosphorylation. According to these criteria ten mutants in the ATPase complex were classified having lesions in the unc A,B region of the chromosome. The first mutant type lacks ATPase activity, but the membrane-integrated part of the complex remains functional (class I). The second mutant type lacks a functional membrane-integrated part, but retains ATPase activity (class II). The third mutant type is shown to be defective in both parts of the ATPase complex (class III).