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.     

The oscillatory network of glycolysis: a model for multiple phase output derived from a single master oscillator

In oscillatory glycolysing yeast cells the reaction rates of 11 enzymic steps are almost completely in phase whereas the concentrations of intermediates fluctuate with highly different phases. Metabolite concentrations depend on the amplitudes of reaction rates even more than on their phases. In this way almost, synchronized enzymes can given rise to a variety of phases, resembling a "master oscillatory" which controls a series of peripheric event with different phases.     

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.     

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.     

Alternative splicing generates two isoforms of the alpha 2 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein which displays developmental heterogeneity in the mammalian central nervous system. Here we describe 2 novel cDNA variants of the rat GlyR alpha 2 subunit and demonstrate that alternative splicing generates these 2 isoforms. The deduced protein sequences (alpha 2A and alpha 2B) exhibit 99% identity with the previously characterized human alpha 2 subunit. In situ hybridization revealed expression of both alpha 2A and alpha 2B mRNAs in the prenatal rat brain, suggesting that these variant proteins may have a role in synaptogenesis. Heterologous expression in Xenopus oocytes showed that the more abundantly expressed alpha 2A subunit forms strychnine-sensitive ion channels which resemble human alpha 2 subunit GlyRs in their electrophysiological properties.     

Mechanistic studies on thrombin catalysis

The kinetic mechanism of the cleavage of four p-nitroanilide (pNA) substrates by human alpha-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates at the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of kcat/Km were bell-shaped for all substrates. The pKa values determined from the pH dependence of kcat/Km for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of kcat/Km for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA and for the binding of the competitive inhibitor N alpha-dansyl-L-arginine-4-methylpiperidine amide. The groups responsible for the observed pKa values were proposed to be His57 and the alpha-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pKa values of 6.7 and 8.6 observed in the pH profile of kcat/Km for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuraxin corresponds to a C-terminal fragment of microtubule-associated protein 5 (MAP5)

From cloned DNA, neuraxin has been identified as a tubulin binding protein of predicted molecular weight of 94 kDa. The deduced sequence of the rat protein exhibits high homology to the C-terminal region of mouse microtubule-associated protein 5 (MAP5). Here, we show that different neuraxin antibodies recognize MAP5, but fail to detect a protein of 94 kDa, in subcellular and microtubular fractions of the rat central nervous system. Furthermore, tubulin binding by neuraxin was found to be dependent on taxol. These data are consistent with neuraxin corresponding to a C-terminal fragment of MAP5 that contains a low-affinity tubulin binding site.     

Primary culture of capillary endothelium from rat brain

To provide an in vitro system for studies of brain capillary function we developed a method for culture of brain capillary endothelial cells. Capillaries were isolated from rat brain and enzymatically treated to remove the basement membrane and contaminating pericytes. Subsequent Percoll gradient centrifugation resulted in a homogeneous population of capillary endothelial cells that attached to a collagen substrate and incorporated [3H]thymidine. Evidence for the endothelial nature of these cells was provided by the presence of Factor VIII antigen and angiotensin converting enzyme activity and by the failure of platelets to adhere to the cell surface. In addition, the cells were joined together by tight junctions. Thus, primary cultures of these cells retained both endothelial and blood-brain barrier features.     

Correlation of chromatin composition with metabolic changes in nuclei of primitive erythroid cells from chicken embryos

Changes in levels of biosynthesis of DNA, RNA, and histones were compared with relative proportions of each histone class during primitive erythropoiesis in embryonic chicks. We confirmed that erythrocyte-specific histone 5 (H5) was substantial in the earliest accessible, erythroblast-enriched stage and that it doubled in relative amount between polychromatic and orthochromatic stages to about 1 mol per 2 mol of each nucleosomal histone, still considerable less than in adult definitive erythrocytes. No other histones changed during primitive erythropoiesis, but the molar proportion of histone 1 (H1) always exceeded that of H5 in these cells, unlike definitive erythrocytes. The increase in content of H5 was accompanied by continued decline in synthesis of the other histones and DNA. The accumulation of H5 during development appears to occur in steps corresponding to the maturation of the primitive and definitive erythroid cell lines. Lysine-rich histones were more easily extracted from nuclei of the erythrosynthesis in whole cells and in isolated nuclei.