Cytogenetic analysis of the L5178Y/TK+/− → TK−/− mouse lymphoma mutagenesis assay system

The L5178Y/TK^+/? → TK^?/? mouse lymphona mutagen assay, which allows selection of forward mutations at the autosomal thymidine kinase (TK) locus, uses a TK^+/? heterozygous cell line, TK^+/? 3.7.2C. Quantitation of colonies of mutant TK^?/? cells in the assay forms the basis for calculations of mutagenic potential of test compounds. We have evaluated the banded karyotypes of the parent TK^+/? heterozygous cell line, as well as homozygous TK^?/? mutants, in order to relate the genetic and morphological properties of mutant colonies. The parent cell line displays karyotype homogeneity, all cells containing normal mouse chromosomes, readily identifiable chromosome rearrangements, and cell line specific marker chromosomes. Mutant TK^?/? colonies of the TK^+/? 3.7.2C cell line form a bimodal frequency distribution of colony sizes for most mutagenic or carcinogenic test substances. Large-colony (λ) TK^?/? mutants with normal growth kinetics appear karyotypically identical within and among clones and with the TK^+/? parental cell line. In contrast, most slow-growing small-colony (σ) TK^?/? mutants have readily recognizable chromosome rearrangements involving chromosome 11, which contains the thymidine kinase gene locus. It is possible that the heritable differences in growth kinetics and resultant colony morphology in λ and σ mutants are related to the type of chromosomal damage sustained. Large-colony mutants receive minimal damage, possibly in the form of point mutations at the TK locus, while small-colony mutants receive damage to other genetic functions coordinately with loss of TK activity, implying gross insult to chromosomal material. It seems likely that λ and σ mutants result from 2 different mutational mechanisms that may be distinguished on the basis of mutant colony morphology.     

The response of murine lymphoma cells — L5178Y S & R — to heat at 44 °C

Two murine lymphoma cell lines L5178YS and L5178YR were exposed to heat at 44 °C. In comparison with other cell lines these were found to be among the most sensitive to the lethal action of heat studied to date. The difference between these cell lines in response to heat was very small when compared to the differences observed with ionizing radiation.The pattern of leakage of radiolabelled leucine at 44 °C was measured in each cell line and small changes compared with the behaviour at 37 °C were observed. No net breakdown or loss of preformed protein was observed during heating nor did any labelled protein leak from the cells.     

Activity of the plant flavanol quercetin in the mouse lymphoma L5178Y TK+/− mutation,DNA single-strand break,and Balb/c 3T3 chemical transformation assays

The activity of quercetin was investigated in (a) the L5178Y TK^+/− mutation assay system, using trifluorothymidine (TFT) as the selection agent; (b) the DNA single-strand break assay in L5178Y cells after the same treatment used for the mutation assay; and (c) the Balb/c 3T3 chemical transformation assay (foci method). Quercetin was active in the TK^+/− mutation assay, increasing the frequency of TFT-resistant colonies from a control value of 37 per 10⁶ viable cells to 355 per 10⁶ viable cells at 20 μg/ml. When S9 was present, the activity was decreased at each concentration tested. As the S9 concentration employed (mg/ml protein) was decreased, the induced mutant frequency increased. DNA single-strand breakage was observed without S9 at 10 μg/ml, using the alkaline elution technique; a maximal rate of elution was reached at 20 μg/ml. In the chemical transformation experiments, transformation just at the level of 0.05% significance (if both intermediate and typical transformed colonies were combined) was observed. The evidence is sufficiently strong that additional attention should be given to its role as a dietary caused of human cancer.     

An evaluation of the L5178Y TK+/− mouse lymophoma forward mutation assay using 42 chemicals

The L5178YTK^+/? mouse lymphoma assay (MLA) has been utilized in several laboratories as a short-term test for chemical-induced forward mutation in cultured mammalian cells. In order to evaluate several technical modifications to the MLA, 42 chemicals representing 9 chemical classes were tested and the results were compared with those published elsewhere as well as with findings in a genetic toxicology test battery currently used in this laboratory. A positive response for the induction of TK^+/? mutants was obtained for 26 chemicals. With the exception of p-aminophenol, all of these compounds were recognized mutagens or carcinogens and were represented of direct-acting and activation-dependent genotoxins. 16 compounds did not induce IK^?/? mutanants and among these were 5 compounds that were considered to be mutagens or carcinogens. A comparison of the results of this study with those published elsewhere revealed a strong agreement among findings for this test irrespective of minor technical variations. It was concluded that th MLA is a useful system for identifying chemical mutagens in mammalian cells and can serve as a valuabel component in a genetic toxicology test battery.     

Genotoxicity of 6 oxime compounds in the Salmonella/mammalian-microsome assay and mouse lymphoma TK+/− assay

To aid in the selection of chemical candidates for in vivo tests, the mutagenicity of 6 oxime compounds was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK^+/− assay. All of the oximes were mutagenic in the mouse lymphoma assay in the absence of exogenous metabolic activation. Acetaldehyde oxime was also mutagenic in the presence of S9 activation. In contrast to these results, a positive response was noted only for 2-(hydroxyimino)-N-phenyl-acetamide oxime in strain TA1535 in the absence of activation in the Salmonella/microsome test.     

The influence of peroxisome proliferator-activated receptor γ(1) during differentiation of mouse embryonic stem cells to neural cells

Peroxisome proliferator activated receptor γ, belongs to PPARs, which exerts various metabolic functions including differentiation process. To testify the importance of PPARγ in neural differentiation of mouse embryonic stem cells (mESCs), its expression level was assessed. Data revealed an elevation in expression level of PPARγ when neural precursors (NPs) are formed upon retinoic acid treatment. Thus, involvement of PPARγ in two stages of neural differentiation of mESCs, during and post-NPs formation was examined by application of its agonist and antagonist. Our results indicated that PPARγ inactivation via treatment with GW9662 during NPs formation, reduced expression of neural precursor and neural (neuronal and astrocytes) markers. However, PPARγ inactivation by antagonist treatment post-NPs formation stage only decreased the expression of mature astrocyte marker (Gfap) suggesting that inactivation of PPARγ by antagonist decreased astrocyte differentiation. Here, we have demonstrated the stage dependent role of PPARγ modulation on neural differentiation of mESCs by retinoic acid treatment for the first time.     

The role of adherent cells in the immunosuppressed state of mouse progeny transplacentally exposed to benzo(α)pyrene

In recent studies, we showed that murine fetal liver cells from progeny exposed to benzo(α)pyrene in utero by intraperitoneal injection of the dam at midpregnancy (12 d) suppressed cell proliferation in an allogeneic mixed lymphocyte response. On the other hand, fetal liver cells from the corn oil (vehicle for the carcinogen)-exposed progeny (control) appeared to enhance proliferation. Suppression or enhancement appeared to be mediated by fetal liver bearing CD8⁺ and Lyt 1⁺ (CD5⁺) cells. Despite these manifestations, the role of third-party cells needs to be considered. As a first premise, adherent cells were targeted as possible third-party cells. To test the role of the adherent cells, liver cells from benzo(α)pyrene-exposed fetuses were treated with ficoll-hypaque, and the interface cells were fractionated through glass wool or nylon wool. It is known that adhering cells, macrophages and B cells, readily attach to glass or nylon wool. The effluent cells and the adherent cells were cultured with syngeneic responder cells and allogeneic stimulator cells in a mixed lymphocyte response. The results showed that benzo(α)pyrene-effluent cells led to the enhancement of proliferation in the mixed lymphocyte response, while benzo(α)pyrene-adherent cells led to suppression. The effluent and adherent cells of corn oil controls did not modify cell proliferation in the mixed lymphocyte response. These data suggest that a third-party cell, reasonably the macrophage or possibly B cells or both, since these are adherent cells, play a decided role in mediating suppression in the benzo(α)pyrene-exposed progeny.     

The history of renalase from amine oxidase to α-NAD(P)H-oxidase/anomerase

Renalase is a recently discovered secretory protein, which plays a certain (still poorly understood) role in regulation of blood pressure. The review summarizes own and literature data on structure and catalytic properties of renalase accumulated since the first publication on this protein (2005). Initial reports on FADdependent amine oxidase activity were not confirmed in independent experiments performed in different laboratories. In addition, proposed amine oxidase activity of circulating extracellular renalase requires the presence of FAD, which has not been detected either in blood or urinary renalase. Moreover, renalase excreted into urine lacks its N-terminal peptide, which is ultimately needed for accommodation of the FAD cofactor. Results of the Aliverti’s group on NAD(P)H binding by renalase and weak diaphorase activity of this enzyme stimulated further studies of renalase as NAD(P)H oxidase catalyzing reaction of catecholamine co-oxidation. However, physiological importance of such extracellular catecholamine-metabolizing activity remains unclear due to existence of much more active enzymatic systems (e.g., neutrophil NAD(P)H oxidase, xanthine oxidase/xanthine) in circulation, which can perform such co-oxidation reactions. Recently α-NAD(P)H oxidase/anomerase activity of renalase, which also promotes oxidative conversion of β-NADH isomers inhibiting activity of NAD-dependent dehydrogenases, has been described. However, its possible contribution to the antihypertensive effect of renalase remains unclear. Thus, the antihypertensive effect of renalase still remains a phenomenon with unclear biochemical mechanim(s) and functions of intracellular and extracellular (circulating) renalases obviously differ.     

Site-selective binding of Zn(II) to metallo-β-lactamase L1 from Stenotrophomonas maltophilia

Extended X-ray absorption fine structure studies of the metallo-β-lactamase L1 from Stenotrophomonas maltophilia containing 1 and 2 equiv of Zn(II) and containing 2 equiv of Zn(II) plus hydrolyzed nitrocefin are presented. The data indicate that the first, catalytically dominant metal ion is bound by L1 at the consensus Zn₁ site. The data further suggest that binding of the first metal helps preorganize the ligands for binding of the second metal ion. The di-Zn enzyme displays a well-defined metal–metal interaction at 3.42 Å. Reaction with the β-lactam antibiotic nitrocefin results in a product-bound species, in which the ring-opened lactam rotates in the active site to present the S1 sulfur atom of nitrocefin to one of the metal ions for coordination. The product bridges the two metal ions, with a concomitant lengthening of the Zn–Zn interaction to 3.62 Å.     

The succession of Staphylinidae (Coleoptera) after 15 years of conversion from conventional to organic farming

The results of a 15 years investigation after the conversion from conventional to organic farming are presented for the beetle family Staphylinidae. The aim of the study was to find general trends in the succession of assemblages, changes in the biodiversity and changes of dispersion for invading or retreating species. The succession of all fields studied are shifted in the same direction, which indicates an overall driving ecological factor after the conversion. However, individual fields showed a different speed within the succession. The number of species remained on the same level in the first 8 years but increased from the eighth to the 15th year. However, the species diversity on the organic fields were still lower than in the field margins. The conventionally farmed fields in 2001 before the conversion of the main study area was compared with adjacent conventionally farmed fields investigated 15 years later. This comparison showed that the species diversity was currently much lower there than 15 years before in the main study area. During the succession single species retreated from fields after the conversion and other species invaded the fields. In particular, species of open habitats and inquilines of mammals invaded the fields. It was concluded that organic farming had benefit effects on biodiversity, but a mosaic with field margins will enhance this effect.     

The demonstration of chloride ions in the “chloride cells” of the gills of eels (Anguilla anguilla L.) adapted to sea water

Summary The ultrastructure of chloride cells in the gills of eels kept in artificial sea water and of a control animal kept in fresh water was studied. In addition to glutaraldehydeosmium tetroxide and simple osmium tetroxide fixation, a special method for the demonstration of chloride ions was used (Komnick, 1962, 1963). Based on the principle of silver chloride precipitation in the presence of chloride ions, the procedure showed positive results in the chloride cells of eels adapted to sea water. Smooth-surfaced tubules of the endoplasmic reticulum contained a material of medium to strong electron density, that was often in communication with the plasma membrane. The same material, always of very high density, was present in the intercellular spaces, thus forming conspicuous lines around cells. The silver precipitate was found very often in large quantities in the pits of chloride cells, having thereby the aspect of a secretory product. However, a direct communication between the system of endoplasmic reticulum tubules containing the silver reaction product with the above-mentioned masses of silver chloride was not demonstrated. Schultz (1958) first published an electron micrograph of these cells. Two features, numerous mitochondria and highly developed agranular endoplasmic reticulum, affirmed by all later investigators, have remained the most important criteria for the identification of chloride cells (Kessel and Beams, 1962; Philpott, 1962; Philpott and Copeland, 1963; Rhodin, 1964; Henrikson and Matoltsy, 1968). Kessel and Beams (1962) and Philpott and Copeland (1963) demonstrated pits, or apical cavities (Oberg, 1967) filled with an amorphous granular substance of medium electron density. (Threadgold and Houston, 1964). These cavities are found in animals adapted to sea water only, and correspond to the excretory vesicles described earlier by Copeland (1948). Philpott and Copeland demonstrated numerous vesicles and tubules in the cytoplasm surrounding the apical cavities and opening into them, possibly contributing to their granular material. Several authors attempted to demonstrate a chloride excretory function of chloride cells with the aid of some histochemical reactions. Copeland (1948), Datta Munshi (1964) and Philpott (1966) used silver techniques     

The systematic value of nuclear genome size for “all” species of Tulipa L. (Liliaceae)

Nuclear genome size, as measured by flow cytometry with propidium iodide, was used to investigate the relationships within the genus Tulipa L. (Liliaceae). More than 400 accessions representing 123 taxa from mainly wild-collected plants were investigated. Most species of Tulipa have the same basic chromosome number, 2n = 2x = 24. However, the somatic DNA 2C value (2C) is shown to range from 32 to 69 pg for the diploids. The largest genome contains roughly 3.4 × 10¹⁰ more base pairs than the smallest and has chromosomes that are more than twice as large. These large differences in the amount of nuclear DNA predict that the hybrids, if any arise, are usually sterile. Depending on the size of the total genome, 1 pg amounts to several thousand genes. Moreover, genome sizes are evaluated here in combination with available morphological, geographical, and molecular data. Therefore, the taxonomy proposed here is not a single-character taxonomy based on genome size alone. The genus Tulipa, as here determined, has 87 species, 29 more than accepted by van Raamsdonk et al. [Acta Hort (ISHS) 430:821–828, 1997], but including 25 species that were not available to them. Of these 87 species, 28 were not seen by Hall (The genus Tulipa, The Royal Horticultural Society, London, 1940) in a living state and placed by him in an addendum. Species of the subgenus Clusianae (Baker) Zonn. differ strongly in nuclear DNA content (DNA 2C value), 32 versus 40–68 pg for all other tulips, and are placed here in a separate subgenus. Also Orithyia, the only group with a style and with only 38–39 pg is placed in a separate subgenus. Therefore, all tulips are attributed to four subgenera, Clusianae (Baker) Zonn., Tulipa, Eriostemones Raamsd., and Orithyia (D. Don) Baker and divided further into 12 sections. Seven of the eight series of section Eichleres (A.D. Hall) Raamsd. are now placed in four sections: (1) section Lanatae (Raamsd.) Zonn., mainly confined to species from the Pamir-Alay and including series Lanatae Raamsd., (2) section Multiflorae (Raamsd.) Zonn. (including series Glabrae Raamsd.), (3) section Vinistriatae (Raamsd.) Zonn. (including series Undulatae Raamsd.), and (4) section Spiranthera Vved. ex Zonn. and Veldk. Triploids, tetraploids, and pentaploids were found in several species. DNA content confirmed the close relationships of the species within the different sections. The rather similar looking and therefore often confused T. armena Boiss. (51.8 pg), T. systola Stapf (56.3 pg), and T. julia K., Koch (61.6 pg) could be clearly distinguished. The same is true for T. biebersteiniana Schult. f. (56.9 pg), T. sylvestris ssp. australis (Link) Pamp. (62.0 pg), and T. primulina Baker (64.6 pg). T. doerfleri Gand. and T. whittalli (Dykes) Hall could be placed as polyploid forms of T. orphanidea Boiss. ex Heldr. On the basis of DNA content, a systematic association between T. julia K. Koch and the triploid T. aleppensis Boiss. and between T. systola Stapf and the triploid T. praecox Tenore was suggested. The new species T. lemmersii Zonn., Peterse, and de Groot is described, and four possible new species are indicated. Genome size as measured by using flow cytometry may conveniently be used to produce systematic data. It is applicable even in the case of dormant bulbs or sterile plants for monitoring the trade in bulbous species.     

Altered sensitivity of cerebellar granule cells to glutamate receptor overactivation in the Cln3(Δex7/8)-knock-in mouse model of juvenile neuronal ceroid lipofuscinosis

The juvenile onset form of neuronal ceroid lipofuscinoses (JNCL) is a recessively inherited lysosomal storage disorder characterized by progressive neurodegeneration. JNCL results from mutations in the CLN3 gene that encodes a lysosomal membrane protein with unknown function. Utilizing a Cln3-knock-out mouse model of JNCL that was created on the 129S6/SvEv genetic background, we have previously demonstrated that CLN3-deficient cerebellar granule cells (CGCs) have a selectively increased sensitivity to AMPA-type glutamate receptor-mediated toxicity. Our recent findings that CGCs from 129S6/SvEv and C57BL/6J wild type (WT) mice have significant differences in glutamate receptor expression and in excitotoxic vulnerability indicated that the genetic background possibly have a strong influence on how glutamate receptor function is dysregulated in CLN3-deficient neurons. Indeed, here we show that in the Cln3(Δex7/8)-knock-in mouse model, that is on the C57BL/6J genetic background, mimics the most frequent mutation observed in JNCL patients and considered a null mutant, the sensitivity of CGCs to both AMPA- and NMDA-type glutamate receptor overactivations is altered. Cultured wild type and Cln3(Δex7/8) CGCs were equally sensitive to AMPA toxicity after 2 or 3 weeks in vitro, whereas the subunit-selective AMPA receptor agonist, CPW-399, induced significantly more cell death in mature, 3-week-old Cln3(Δex7/8) cultures. NMDA receptor-mediated toxicity changed during in vitro development: Cln3(Δex7/8) CGCs were less sensitive to high concentration of NMDA after 2 weeks in culture but became more vulnerable than their WT counterparts after 3 weeks in vitro. Abnormally altered glutamate receptor function in the cerebellum may result in motor deficits, and we confirmed that 7-week-old Cln3(Δex7/8) mice, similarly to Cln3-knock-out mice, have a motor coordination deficit as measured by an accelerating rotarod. Our results demonstrate altered glutamate receptor function in Cln3(Δex7/8) neurons and suggest that both AMPA and NMDA receptors are potential therapeutic targets in JNCL.     

Immunological characterization of multipotent mesenchymal stromal cells—The International Society for Cellular Therapy (ISCT) working proposal

Cultured mesenchymal stromal cells (MSCs) possess immune regulatory properties and are already used for clinical purposes, although preclinical data (both in vitro and in vivo in animal models) are not always homogeneous and unequivocal. However, the various MSC-based clinical approaches to treat immunological diseases would be significantly validated and strengthened by using standardized immune assays aimed at obtaining shared, reproducible and consistent data. Thus, the MSC Committee of the International Society for Cellular Therapy has decided to put forward for general discussion a working proposal for a standardized approach based on a critical view of literature data.     

Purification and characterization of a soluble β-1,4-glucan from bean (Phaseolus vulgaris L.)-cultured cells dehabituated to dichlobenil

Bean cells habituated to grow in the presence of dichlobenil exhibited reduced cellulose and hemicellulose content and an increase in pectic polysaccharides. Furthermore, following the extraction of pectins and hemicelluloses, a large amount of neutral sugars was released. These sugars were found to be part of a soluble β-1,4-glucan in a preliminary characterization, as reported by Encina et al. (Physiol Plant 114:182–191, 2002). When habituated cells were subcultured in the absence of the herbicide (dehabituated cells), the release of neutral sugars after the extraction of pectins and hemicelluloses was maintained. In this study, we have isolated a soluble β-1,4-glucan from dehabituated cells by sonication of the wall residue (cellulose fraction) remaining after fractionation. Gel filtration chromatography revealed that its average molecular size was 14 kDa. Digestion of the sample with endocellulase revealed the presence of cellobiose, cellotriose, and cellotetraose. Methylation analysis showed that 4-linked glucose was the most abundant sugar residue, but 4,6-linked glucose, terminal arabinose and 4-linked galactose for xyloglucan, and arabinogalactan were also identified. NMR analysis showed that this 1,4-glucan may be composed of various kinds of substitutions along the glucan backbone together with acetyl groups linked to the OH group of sugar residues. Thus, despite its relatively high molecular mass, the β-glucan remains soluble because of its unique configuration. This is the first time that a glucan with such characteristics has been isolated and described. The discovery of new molecules, as this β-glucan with unique features, may help understand the composition and arrangement of the polymers within plant cell walls, contributing to a better understanding of this complex structure.