An investigation of dye reduction by food-borne bacteria

S.A. LEAROYD, R.G. KROLL AND C.F. THURSTON. 1992. The rates of reduction of seven redox dyes by 13 bacterial strains were measured and found to vary greatly between different bacterium/dye combinations. Phenazine ethosulphate and toluidine blue were the most rapidly reduced dyes by the majority of bacteria and resorufin and 2-hydroxy-1,4-naphthoquinone were reduced slowly, if at all. There was also considerable variation in the rates of reduction with any single dye/organism combination. Glucose stimulated the rates of endogenous dye reduction in about half of the organisms. For Bacillus cereus, Pseudomonas fluorescens and Escherichia coli , dye reduction was stimulated by a range of exogenous substrates but lactose, the primary available carbon and energy source in milk, had little effect. In Lactococcus lactis , dye reduction was stimulated by sugars but not by organic acids. Oxygen successfully competed with dye reduction in organisms containing respiratory chains, but with membrane fractions, dye reduction was more rapid than oxygen consumption. All the organisms showed little cytosolic dye reduction, except L. lactis which showed substantial rates of reduction of some dyes by this fraction. With the membrane fraction of E. coli and Ps. fluorescens , cyanide inhibited NADH and succinate-dependent dye reduction, Antimycin A inhibited lactate and succinate and rotenone had no significant effect, but inhibition was not always observed with membrane from both organisms.     

The Crustacea of some chalk streams in southern England

Records of Crustacea from chalk streams in southern England are described. Classification of sites suggested that flow regime was an important influence on the fauna and the distribution of individual species are discussed in this respect.     

The latex agglutination test versus counterimmunoelectrophoresis for rapid diagnosis of bacterial meningitis

A modified latex agglutination (LA) test was compared with Gram-staining and counterimmunoelectrophoresis (CIE) for the rapid detection in the cerebrospinal fluid (CSF) of antigen to Haemophilus influenzae type b, Neisseria meningitidis groups A, B and C, Escherichia coli K1, Streptococcus pneumoniae and group B streptococci, seven frequent causes of bacterial meningitis in children. Of 50 CSF samples from patients with culture-proven bacterial meningitis 90% were correctly shown by the LA test to contain antigen of the responsible organism. Gram-staining revealed organisms in 80% of 45 of these samples. In 75% of the 40 samples that were of sufficient volume for CIE, positive results for the appropriate antigen were obtained. The concentration of antigen detected in the CSF by the LA test varied from undetectable to 800 000 ng/ml. Patients with a high concentration (more than 2000 ng/ml or a positive result at dilutions of CSF over 1/8) were significantly more likely to have a poor response to therapy (two died and two had persistent pleocytosis or bacteria in the CSF) than patients with a lower concentration (4/16 v. 0/18, P < 0.05). After appropriate therapy was begun the concentration of antigen fell dramatically, but measurable amounts of antigen persisted in the CSF for up to 6 days. The LA test detected bacterial antigen at concentrations 2 to 70 times below the lower limit detected by CIE. In seven additional patients who had received antibiotics before lumbar puncture was performed the LA test detected antigen from meningitis-causing bacteria even though cultures of the CSF were sterile. In another 145 patients who did not have meningitis the results of the LA test were negative. The LA test, done as described in this article, is easier to perform than CIE and should be a useful addition to the diagnostic tests carried out on the CSF of any patient suspected of having meningitis.     

Binding of epithelial cells to lectin-coated surfaces

Summary Epithelial cells may relate to their basement membrane substrates via lectin-like interactions. In a model system for study of this type of interaction, lectin-coated bacteriological plastic petri dishes were presented as substrates for epithelial cell adhesion. Of 21 lectins tested by mixed agglutination against two epithelial cell types, Madin-Darby canine kidney (MDCK), and human embryonic kidney cells (HEK), nine gave less than 5% rosettes and 12 gave 5 to 50% rosettes. Wheat germ agglutinin (WGA) andGeodia cydonium lectin gave the highest percentage of rosettes. Wheat germ agglutinin was readily adsorbed to plastic surfaces and maintained specificity in binding interactions. Both MDCK and HEK cells attached as well to WGA coated petri dishes as to conventional tissue culture dishes. Furthermore, both spread over the lectin-coated surfaces. The MDCK cells grew to confluence and could be subcultured and maintained indefinitely on such surfaces, although WGA in solution was toxic to the cells in concentrations as low as 0.1 to 1.0 μg/ml. Cell attachment to WGA coated dishes was blocked by cycloheximide only if the cells had been preincubated with the inhibitor for several hours. Cell attachment was not inhibited by pretreatment of cells with neuraminidase. Precoating cells with WGA blocked binding to both WGA-coated surfaces and untreated tissue culture dishes. Cells attached to WGA-coated dishes could not be readily dislodged by trypsin-EDTA for the first 2 h after subculture. By 4 h, attachment was again trypsin sensitive, suggesting that the cells synthesized a trypsin-sensitive material that was laid down between the cell surface and the WGA-coated dish. Regeneration of trypsin sensitivity was not blocked by cycloheximide. This work was supported by Research Grant AG01986 from the National Institutes of Health, Bethesda, Maryland.     

Contraction of guinea pig uterus by synthetic leukotrienes

Synthetic leukotrienes (LT) C₄ and D₄ elicited concentration-dependent contractions of the guinea pig uterus between 10^?8-10^?6M, whereas LTE₄ appeared 1000-fold weaker. The potencies of LTC₄ and LTD₄ were similar to that of acetylcholine and PGF_2α but weaker than that of PGE₂. The maximal contractions elicited by LTC₄ and LTD₄ were 66.0 ± 2.1% and 63.8 ± 4.6% that elicited by acetylcholine. FPL 55712 (10^?5M) antagonized the uterine contractile activity of LTD₄, while meclofenamic acid at 10^?5M but not at 10^?6M also antagonized the LTD₄-induced contration. Radioimmunoassay of the uterine tissue bathing fluid following LTD₄ indicated the variable presence of low concentrations of PGE₂, PGF_2α and TXB₂. These results demonstrate the LTC₄ and LTD₄ possess significant uterine contractile activity, which may only partially be mediated indirectly via prostaglandin products.     

Active sulfate absorption in rabbit ileum: Dependence on sodium and chloride and effects of agents that alter chloride transport

Summary Unidirectional fluxes of³⁵SO₄ across and into rabbit ileal epithelium were measured under short-circuit conditions, mostly at a medium SO₄ concentration of 2.4mm. Unidirectional mucosa (m)-to-serosa (s) ands-to-m fluxes (J _ms,J _sm) were 0.456 and 0.067 moles hr^–1 cm^–2, respectively.J _ms was 2.7 times higher in distal ileum than in mid-jejunum. Ouabain abolished net SO₄ transport (J _net) by reducingJ _ms. Epinephrine, a stimulus of Cl absorption, had no effect on SO₄ fluxes. Theophylline, a stimulus of Cl secretion, reducedJ _ms without affectingJ _sm, causing a 33% reduction inJ _net. Other secretory stimuli (8-Br-cAMP, heat-stable enterotoxin, Ca-ionophore A23187) had similar effects. Replacement of all Cl with gluconate markedly reducedJ _net through both a decrease inJ _ms and an increase inJ _sm. The anion-exchange inhibitor, 4-acetoamido-4-isothiocyano-2,2-sulfonic acid stilbene (SITS), when added to the serosal side, reducedJ _ms by 94%, nearly abolishingJ _net. SITS also decreasedJ _sm by 75%. Mucosal SITS (50 m) was ineffective. 4,4-diisothiocyano-2,2-sulfonic acid stilbene (DIDS) had effects similar to SITS but was less potent. Measurements of initial rates of epithelial uptake from the luminal side (J _me) revealed the following: (1)J _me is a saturable function of medium concentration with aV _max of 0.94 moles hr^–1 cm^–2 and aK _1/2 of 1.3mm; (2) replacing all Na with choline abolishedJ _me; (3) replacing all Cl with gluconate increasedJ _me by 40%; (4) serosal SITS had no effect onJ _me; and (5) stimuli of Cl secretion had no effect onJ _me or increased it slightly. Determination of cell SO₄ with³⁵SO₄ indicated that, at steady-state, the average mucosal concentration is 1.1 mmoles per liter cell water, less than half the medium concentration. Cell SO₄ was increased to 3.0mm by adding SITS to the serosal side. Despite net transport rates greater than 1.4 Eq hr^–1 cm^–2, neither addition of SO₄ to the SO₄-free medium nor addition of SITS to SO₄-containing medium altered short-circuit current. The results suggest that (1) ileal SO₄ absorption consists of Na-coupled influx (symport) across the brush border and Cl-coupled efflux (antiport) across the basolateral membrane; (2) the overall process is electrically neutral; (3) the medium-to-cell Cl concentration difference may provide part of the driving force for net SO₄ absorption; and (4) since agents affecting Cl fluxes (both absorptive and secretory) have little effect on SO₄ fluxes, the mechanisms for their transcellular transports are under separate regulation.     

Absence of nucleolar dominance in mouse-human heterokaryons

Autoradiographic analysis of [³H]uridine incorporation 48 h after polyethylene glycol-mediated cell fusion indicates that nucleolar RNA synthesis persists in both human and mouse nuclei in interspecific heterokaryons. The absence of nucleolar dominance in heterokaryons has been confirmed by zinc-dithizone nucleolus-specific staining, and is true even when there are considerably more nuclei of one species than of the other in the heterokaryon. Studies of actinomycin D-induced nucleolar segregation indicate that the zinc-binding proteins responsible for zinc-dithizone staining are located in a different nucleolar component than the protein responsible for silver staining.     

Spontaneous cell hybridization of somatic cells present in sperm suspensions

Electron microscopic evidence suggests that sperm can be spontaneously incorporated by cultured cells but cytogenetic and biochemical evidence indicate that sperm do not introduce new genes into such cells with detectable frequency. Sperm suspensions from mouse or Chinese hamster epididymis or human semen were added to cultures of RAG, a mouse cell line which dies in HAT medium because of HPRT deficiency. In EMs, sperm appeared to be readily phagocytized and degraded by the cells. When sperm-treated cultures were transferred to HAT medium resistant clones arose at a frequency of about 10⁻⁶, or at least 25× the reversion rate of RAG. Most HAT-resistant clones had HPRT activity which migrated electrophoretically like HPRT of the sperm donor species, though one was apparently a spontaneous RAG revertant. Most HAT-resistant clones had some chromosomes of the sperm donor species. In human sperm× RAG clones, the array of human chromosomes suggested that the human parent had been diploid rather than haploid; some cells contained both homologues of a polymorphic pair and some contained both X and Y. Furthermore, some sperm suspensions plated alone into flasks generated colonies, thus revealing the presence of low numbers of viable somatic cells. Presence of contaminating somatic cells in a sperm suspension was correlated with ability to induce HAT-resistant colonies when the suspension was added to RAG cells. Taken together, the data suggest that correction of the HPRT deficiency of RAG by sperm suspensions occurs at very low frequency and is probably due to efficient spontaneous fusion of low numbers of contaminating somatic cells with RAG cells.     

Potent inhibition of microRNA in vivo without degradation

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.