Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

The role of sediment type in growth and fecundity of mud snails (Hydrobiidae)

Summary We test the hypothesis that body size and population density of the deposit-feeding gastropod, Hydrobia truncata, are greater in muddy than in sandy habitats as a result of faster growth on fine- compared to coarse-grained sediments. We refute this hypothesis using a combination of field measurements and laboratory experiments. Three out of three populations tested had higher maximal growth rates and two of three populations approached their asymptotic size more quickly on sand than on silt-clay fractions of natural sediment. Growth decreased with increasing snail density and was as high or higher on sand as on silt-clay at all densities. Two populations were more fecund on sand than on silt-clay, and fecundity of the third population was not affected by sediment type. We show that the smaller body sizes observed in snails from the sandiest habitat result from late recruitment of these snails, relative to the other populations.     

Plant Regeneration from Isolated Microspore of Indica Rice

Isolated microspores of rice (Oryza saliva L.) cultivars, IR36and IR43, belonging to the recalcitrant indica subspecies werecultured. Two types of microspores were observed after isolationfrom the fresh anthers and from pre-cultured anthers—onetype consisted of vacuolated, larger-sized grains, while theother was composed of microspores of smaller sizes with densecytoplasm. Within few days in culture, all the smaller sizedgrains were dead, and only the large grains were viable andproduced pollen embryos. After 30 days from culture, microcalliwere transferred to semisolid modified Murashige and Skoog mediumcontaining 1 mg/liter each of kinetin and naphthaleneaceticacid and kept under continuous light at 25?C. IR36 showed onlycell division while IR43 gave 32 green plants from these experiments. (Received January 18, 1990; Accepted July 4, 1990)     

Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH).

Herein we report the results of the first major prospective study directly comparing aneuploidy detection by fluorescence in situ hybridization of interphase nuclei with the results obtained by cytogenetic analysis. We constructed probes derived from specific subregions of human chromosomes 21, 18, 13, X, and Y that give a single copy-like signal when used in conjunction with suppression hybridization. A total of 526 independent amniotic fluid samples were analyzed in a blind fashion. All five probes were analyzed on 117 samples, while subsets of these five probes were used on the remaining samples (because of insufficient sample size), for a total of over 900 autosomal hybridization reactions and over 400 sex chromosome hybridization reactions. In this blind series, 21 of 21 abnormal samples were correctly identified. The remaining samples were correctly classified as disomic for these five chromosomes. The combination of chromosome-specific probe sets composed primarily of cosmid contigs and optimized hybridization/detection allowed accurate chromosome enumeration in uncultured human amniotic fluid cells, consistent with the results obtained by traditional cytogenetic analysis.     

Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungusPleurotus eryngii

Summary Production of extracellular hydrogen peroxide by fungal oxidases is been investigated as a requirement for lignin degradation. Aryl-alcohol oxidase activity is described in extracellular liquid and mycelium ofPleurotus eryngii and studied under non-limiting nitrogen conditions. This aryl-alcohol oxidase catalyses conversion of primary aromatic alcohols to the corresponding aldehydes and H₂O₂, showing no activity with aliphatic and secondary aromatic alcohols. The enzyme is stable at pH 4.0–9.0, has maximal activity at 45°–50°C and pH 6.0–6.5, is inhibited by Ag⁺, Pb²⁺ and NaN₃, and has aK _m of 1.2 mM using veratryl alcohol as substrate. A single protein band with aryl-alcohol oxidase activity was found in zymograms of extracellular and intracellular crude enzyme preparations fromP. eryngii.     

Heterotrophic bacteria, activity and bacterial diversity in two coastal lagoons as detected by culture and 16S rRNA genes PCR amplification and partial sequencing

Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prevost Lagoon is more eutrophic than Arcachon Bay. Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allow the determination of phylogenetic relationships among members of microbial communities in natural ecosystems without the need for cultivation. Analysis of partial 16S rRNA gene sequences obtained from Stations A and 11 revealed that, in both environments, a relatively large number of clones related to Cytophaga/FlexibacterBacteroides as well as to -Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied. In fact for most clones maximum similarity was below 95% for the nucleotide series sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Eubacteria were obtained from Prevost Lagoon, however only five branches were represented by the data from Arcachon. These findings indicate a higher bacterial diversity in Prévost Lagoon.