The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels.

Purified preparations of the inhibitory glycine receptor (GlyR) contain alpha and beta subunits, which share homologous primary structures and a common transmembrane topology with other members of the ligand-gated ion channel superfamily. Here, a beta subunit-specific antiserum was shown to precipitate the [3H]strychnine binding sites localized on alpha subunits from membrane extracts of both rat spinal cord and mammalian cells co-transfected with alpha and beta cDNAs. Further, inhibition of alpha homo-oligomeric GlyRs by picrotoxinin, a non-competitive blocker of ion flow, was reduced 50- to 200-fold for alpha/beta hetero-oligomeric receptors generated by cotransfection. Site-directed mutagenesis identified residues within the second predicted transmembrane segment (M2) of the beta subunit as major determinants of picrotoxinin resistance. These data implicate the M2 segment in blocker binding to and lining of the GlyR chloride channel.     

Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane.

Synaptophysin, a mol. wt 38 000 glycopolypeptide of the synaptic vesicle membrane, was solubilized using Triton X-100 and purified by immunoaffinity or ion-exchange chromatography. From gel permeation and sucrose-density centrifugation in H2O/D2O, a Stokes radius of 7.3 nm, a partial specific volume of 0.830 and a total mol. wt of 119 000 were calculated for the native protein. Cross-linking of synaptic vesicles with glutaraldehyde, dimethylsuberimidate, or Cu2+ -o-phenantroline, resulted in the formation of a mol. wt 76 kd dimer of synaptophysin. Crosslinking of the purified protein in addition produced tri- and tetrameric adducts of the polypeptide. Native synaptophysin thus is a homooligomeric protein. Synaptophysin is N-glycosylated, since cultivation of the rat phaeochromocytoma cell line PC12 in the presence of tunicamycin reduced its mol. wt by about 6 kd. Upon transfer to nitrocellulose and incubation with 45Ca2+, synaptophysin behaved as one of the major calcium-binding proteins of the synaptic vesicle membrane. Pronase treatment of intact synaptic vesicles abolished this 45Ca2+ binding indicating that the Ca2+ binding site of synaptophysin must reside on a cytoplasmic domain of the transmembrane polypeptide. Based on these data, we propose that synaptophysin may play an important role in Ca2+-dependent neurotransmitter release.     

Use of flow cytometry in industrial microbiology for strain improvement programs

A flow cytometry (FCM) system was chosen to analyze and sort microbiological samples, e.g., bacteria, bacterial spores, yeasts, and fungal spores, without major changes in the commercially available state. The system was further improved by addition of a stepping motor-driven scanning table that accepts standard petri dishes or microtiter plates. The electronics of the sorting system were changed to enable the sorter to deliver only one particle at a time, working in a "handshake" mode with the scanning table. Appropriate parameters, depending on the biological material and including all fluorescent stains that do not impair growth and productivity of cells were chosen to sort distinct bioparticles under aseptic conditions and to clone colonies or cultures out of them. A mutagenized sample of spores entering the germination cycle can be followed and thus provide a means to pick only viable growing cells despite the killing effect of the mutagen. One example of a typical strain improvement is illustrated. From a spore suspension of Rhizopus arrhizus, a subpopulation of morphologically different spores comprising about 5-10% of the whole population was cloned. From approximately 8,000 clones, 10 were isolated that produced approximately five- to six-fold the amount of fungal lipase activity, compared to the original strain or to reisolated clones from the mean population of clones.     

Signal analysis of slit-scan contour data.

As a method for the preselection of alarms in gynecological cell samples, the Battelle Cytophotometry Research Group uses the slit-scan technique to obtain various cell parameters, such as the N/C ratio and the relative DNA content, from fluorescently stained cells, which are aligned one-dimensionally in the tape system designed at Battelle. The system developed at Battelle Institute analyzes all signals that exceed the background noise. As the first step in processing the slit-scan data, several threshold levels permit the separation of various artifacts. In subsequent steps, the nuclear peak is recognized, the nuclear boundaries are calculated, and seven cell parameters are determined. For the alarm detection at present only one parameter, DNA fluorescence, is used for these determinations. Visual assignment of these data to definite objects on the tape makes it possible to obtain frequency distributions of: (a) all recorded objects within the sample on the tape; (b) all signals that are classified as cells; and (c) all types of objects that preferentially cause alarms.     

a-Factor from Saccharomyces cerevisiae: partial characterization of a mating hormone produced by cells of mating type a.

Conjugation between haploid cells of Saccharomyces cerevisiae is mediated through the action of diffusible mating hormones, two of which have been designated as a-factor and alpha-factor. Partially purified fractions exhibiting a-factor activity have been obtained from culture filtrates of a cells by ultrafiltration, ion-exchange chromatography, and gel filtration. The a-factor preparations specifically caused both G1 arrest and morphological alterations in cells of alpha-mating type, whereas a cells, a/alpha diploids, and nonmating alpha mutants were not affected. The a-factor activity was found in the culture filtrates of all a strains tested, but not in filtrates of alpha or a/alpha cell cultures. The hormone is sensitive to various proteases, showing that it is associated with a peptide or protein. Gel filtration studies suggest an apparent molecular weight greater than 600,000; however, this result may be due to aggregation with carbohydrate present in the preparations. Although the biological activities of a-factor are analogous to those described previously for alpha-factor, the chemical properties of these two hormones appear to be quite different.