In vitro stimulation by tumour cell media of [3H]-thymidine incorporation by mouse spleen lymphocytes

Mouse spleen lymphocyte (SL) cells show a three to four-fold increase in [³H]-thymidine incorporation when incubated in tumour cell media, or in media containing tumour cell cytosol. Agarose gel chromatography of both [³H]-thymidine-labelled tumour cell media and cytosol shows a sharp peak of DNA-associated material eluting at about 60 kDa. This DNA-associated material is imported rapidly and efficiently by SL cells and is recoverable from their cytosol. The stimulating effect on SL cell thymidine incorporation resides primarily, if not exclusively, in this extruded/cytosolic 60 kDa DNA material. Tumour cells incubated in media containing normal or liver, but not tumour, cytosol show a reduced rate of [³H]-thymidine incorporation, indicating competition between normal and tumour associated DNA complexes. The results indicate that such cell-extruded DNA complexes may transmit ‘genetic messages’ to other cells, and are discussed in terms of interactions in the tumour-bearing host. © 1997 John Wiley & Sons, Ltd.     

Multiple Deletions of the Osmolyte Transporters BetL, Gbu, and OpuC of Listeria monocytogenes Affect Virulence and Growth at High Osmolarity

The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the food environment and, after ingestion, within the animal host. Growth at high salt concentrations is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We characterized L. monocytogenes LO28 strains with single, double, and triple deletions in the osmolyte transport systems BetL, Gbu, and OpuC. When single deletion mutants were tested, Gbu was found to have the most drastic effect on the rate of growth in brain heart infusion (BHI) broth with 6% added NaCl. The highest reduction in growth rate was found for the triple mutant LO28BCG (ΔbetL ΔopuC Δgbu), although the mutant was still capable of growth under these adverse conditions. In addition, we analyzed the growth and survival of this triple mutant in an animal (murine) model. LO28BCG showed a significant reduction in its ability to cause systemic infection following peroral coinoculation with the wild-type parent. Altering OpuC alone resulted in similar effects (R. D. Sleator, J. Wouters, C. G. M. Gahan, T. Abee, and C. Hill, Appl. Environ. Microbiol. 67:2692-2698, 2001), leading to the assumption that OpuC may play an important role in listerial pathogenesis. Analysis of the accumulation of osmolytes revealed that betaine is accumulated up to 300 μmol/g (dry weight) when grown in BHI broth plus 6% NaCl whereas no carnitine accumulation could be detected. Radiolabeled-betaine uptake studies revealed an inability of BGSOE (ΔbetL Δgbu) and LO28BCG to transport betaine. Indeed, for LO28BCG, no accumulated betaine was found, but carnitine was accumulated in this strain up to 600 μmol/g (dry weight) of cells, indicating the presence of a possible fourth osmolyte transporter.     

Circulating nucleic acids: possible inherited effects

The presence of circulating nucleic acids in man, animals and plants is well documented. It is clear that such nucleic acids can not only circulate freely within an organism, but can also enter cells when their biology may be changed either epigenetically or genetically. Evidence is presented concerning a possible influence of these nucleic acid fragments on the genetics of the F1 generation of man, animals and plants. The data presented also offer a mechanism by which the incorporation of horizontally transferred genes between organisms may be achieved. The role that circulating nucleic acids might play in modifying the F1 generation and possibly the evolutionary process is considered. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 931–948.     

Glycolytic activity in embryos of Pisum sativum and of non-dormant or dormant seeds of Avena sativa L. expressed through activities of PFK and PPi-PFK

A quantitative cytochemical assay for PPi-PFK activity in the presence of Fru-2,6-P2 is described along with its application to determine levels of activity in embryos of Pisum sativum and Avena sativa. The activity of ATP-PFK has also been studied in parallel as have PFK activities during the switch from dormant to non-dormant embryos in Avena sativa. PPi-PFK activity has been demonstrated in all tissues of Pisum sativum embryos and of Avena sativa embryos including the scutellum and the aleurone layers. The PPi-PFK activity was greater than that of ATP-PFK in both dormant and non-dormant seeds though with only marginally more activity in the dormant as opposed to the non-dormant state.     

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

Abstract  

The purple acid phosphatases (PAPs) are the only binuclear metallohydrolases where the necessity for a heterovalent active site [Fe(III)–M(II) (M is Fe, Zn or Mn)] for catalysis has been established. The paradigm for the construction of PAP biomimetics, both structural and functional, is that the ligands possess characteristics which mimic those of the donor sites of the metalloenzyme and permit discrimination between trivalent and divalent metal ions. The donor atom set of the ligand 2-((2-hydroxy-5-methyl-3-((pyridin-2-ylmethylamino)methyl)benzyl)(2-hydroxybenzyl)amino)acetic acid (H₃HPBA) mimics that of the active site of PAP although the iron(III) complex of this ligand has been characterized as the tetramer [Fe₄(HPBA)₂(μ-CH₃COO)₂(μ-O)(μ-OH)(OH₂)₂]ClO₄·5H₂O. The phosphoesterase-like activity of the complex in 1:1 acetonitrile/water has now been investigated using the substrate 2,4-bis(dinitrophenyl)phosphate. The pH dependence of the catalytic rate revealed a non-symmetric bell-shaped profile, with a finite but non-zero rate at high pH. Unlike the traditional approach usually employed to analyse these bell-shaped profiles, the approach used here involved incorporating additional species which contribute to the overall activity. Employing this approach, we show that the complex has a k _cat of 1.6 (±0.2) × 10⁻³ s⁻¹, three kinetically relevant pK _a values of 5.3, 6.2 and 8.4, with K _M of 7.4 ± 0.6 mM. The kinetic parameters are similar to those reported for heterovalent PAP biomimetics. Additionally, it is observed that, unlike the enzyme, the oxidation state is not the determining factor for catalytic activity.     

Factors affecting survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Listeria innocua</Emphasis> in soil samples

We investigated the ability of several strains of L. monocytogenes and Listeria innocua strains to survive in local soil samples in vitro. Survival of three L. monocytogenes strains, EGDe, CD83, and CD1038, and three L. innocua strains, CLIP, FH2117, FH2152, was monitored in soil samples by direct enumeration of colony-forming units on selective agar. The study did not demonstrate any species-specific difference in soil survival, and all Listeria strains exhibited a marked decline in numbers over time. Bioluminescence imaging approaches to detect lux-tagged strains in soil proved largely ineffective, most likely due to the reduced metabolic activity of strains in this environment. We investigated the influence of specific factors including the presence of a background microbiota, growth temperature, moisture and strain motility upon persistence in this environment. A sequenced L. monocytogenes strain, EGDe, was capable of active growth in sterile soil yet exhibited a decline in the presence of the normal soil microbiota. Furthermore, greater survival was seen at lower incubation temperatures in normal soil. Finally, we demonstrated a direct correlation between motility and survival of L. monocytogenes in soil with highly motile L. monocytogenes strains exhibiting greater soil survival than non-motile mutants.