Acid Tolerance of Streptococcus macedonicus as Assessed by Flow Cytometry and Single-Cell Sorting

An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC₄(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.     

Detection, determination, and metabolism in vitro of gangliosides in mammalian sympathetic ganglia

Abstract— Sympathetic ganglia of the rat and cat were examined for the occurrence and distribution of gangliosides. Each rat superior cervical ganglion contained 0.3 nmol of ganglioside-sialic acid. Extracts of cat superior cervical and nodose ganglia were chromatographed on silica gel thin-layer plates. The resulting patterns suggested that similar distributions of multiple forms of gangliosides occur in these two tissues, with the fast-moving gangliosides predominating. The metabolic activity of gangliosides was also investigated in rat superior cervical ganglia in vitro. Evidence was obtained that ¹⁴C from [U-¹⁴C]glucose, [U-¹⁴C]pyruvate, and [U-¹⁴C]glucosamine was incorporated into the gangliosides.     

Use of Miracil D to Suppress Bacterial Ribonucleic Acid and Protein Synthesis During Bacteriophage MS2 Infection

Under certain culture conditions, Miracil (35 mug/ml) halts the growth of uninfected Escherichia coli. Cellular ribonucleic acid (RNA) synthesis is almost completely suppressed, whereas deoxyribonucleic acid and protein synthesis are inhibited to a lesser extent. When the drug is added to host bacteria prior to infection with bacteriophage MS2, the phage adsorb to the cells, but penetration of the viral RNA is inhibited. Penetration may be achieved without further viral development by infection in the presence of chloramphenicol. If the bacteria are infected with MS2 in the presence of chloramphenicol, subsequently washed to remove the chloramphenicol, and then treated with Miracil at any time between 0 and 20 min postinfection, a second viral function is inhibited and the yield of progeny phage is reduced. Addition of the drug after 20 min postinfection does not inhibit the infection process. When Miracil is present from early times in infection, only a limited synthesis of both double- and single-stranded virus-specific RNA is observed. The viral RNA species thus produced do not appear to differ from those made in the absence of the drug. A comparison of the activities of the viral RNA synthetase produced during the course of infection in the presence and in the absence of Miracil suggests that a possible cause of the inhibition is the synthesis of an unstable enzyme in the presence of the drug.     

The effect of paraquat on flax cotyledon leaves: Changes in fine structure

Summary Changes in the fine structure of flax (Linum usitatissimum) cotyledon leaf cells treated with the herbicide paraquat (1,1-dimethyl-4,4-bipyridylium ion.) were investigated. After 6 h treatment under constant illumination, tonoplast breakdown was evident. This was followed by a rapid and progressive deterioration of cell contents including the disruption of mitochondria, and the breakdown of chloroplast thylakoid membrane structure with the accumulation of osmiophilic plastoglobuli. There was no apparent deterioration after 18 h paraquat treatment in darkness but by 30 h there was a limited breakdown of chloroplast membranes.     

Animal collagenases: specificity of action, and structures of the substrate cleavage site

Two separable hormone entities have been found by exploratory purifications and assay for release of growth hormone (GH) by radioimmunoassay. In recognition of frequent multiple activities of peptide hormones, these two hormonal entities are provisionally designated factors A-GHRH and B-GHRH until they are chemically characterized and their dominant functionality clarified. The A- and B-GHRH designations merely define the assay guiding isolation. Factor A-GHRH was found by filtration on Bio-Gel P-2, and purified over Sephadex G-25 in two partition chromatographic systems, and by Sephadex LH-20. The two partitions and stage LH-20 also differentiated the two active entities. Fractions of A-GHRH were active at 100–200 μg. Factor A-GHRH is inhibited by somatostatin.     

Collagenase in carcinoma cells

Collagenolytic enzyme activity has been found in high levels in homogenates of a tumor (ascitic V₂ carcinoma) implanted in rabbit muscle. Unlike other mammalian collagenases, this enzyme was not actively synthesized and released by primary cultures of the tumor. Most of the collagenolytic activity in tumor homogenates was found to sediment at 1000–4000 × g along with 5′-nucleotidase activity indicating that it might be bound to a membraneous component of the cell.