Dinuclear silver(I) complexes of 24-membered bibracchial tetraimine Schiff base macrocycles derived from 2,5-diformylthiophene

The cyclocondensation of 2,5-diformylthiophene and the amines N,N-bis-(2-aminoethyl)-2-phenylethylamine, N,N-bis-(2aminoethyl)-t-butyl-amine and N,N-bis-(2-aminoethyl)-t-butyl-amine in the presence of silver(I) salts yields homodinuclear bibracchial tetraimine Schiff base macrocyclic complexes. The structures of two such complexes are also reported. The complex Ag₂L⁴(NO₃)(PF₆) (2) crystallises in the triclinic space group , No. 2) and has unit-cell dimensions a = 12.834(6), B = 13.183(6), C = 14.588(7) Å, = 64.86(4), β = 79.77(4), γ = 69.44(3)° with Z = 2; there is a monodentate and singly bridging nitrate anion present and the Ag---Ag separation is 4.161 Å. The complex Ag₂L⁴(CH₃CN)₂(BF₄)₂·CH₃CN (9) crystallises in the triclinic space group , No. 2) and has unit-cell dimensions a = 9.297(4), B = 12.985(3), C = 21.770(5) Å, = 91.570(10), β = 92.33(3), γ = 97.92(3) ° with Z = 2; there is a strongly bonded acetonitrile molecule coordinated to each silver atom and the Ag---Ag separation is 4.920 Å.     

Genetic evidence for a complex of species within Rugopharynx australis (Mönnig, 1926) (Nematoda: Stronglyloidea) from macropodid marsupials

An electrophoretic analysis using 17 enzyme loci was carried out on specimens of the gastric nematode of macropodid marsupials, Rugopharynx australis (Mönnig, 1926), collected from Macropus eugenii (Desmarest), M. fuliginosus (Desmarest), M. giganteus Shaw, M. robustus Gould, M. rufogriseus (Desmarest), M. rufus (Desmarest), Thylogale billardierii (Desmarest) and Wallabia bicolor (Desmarest) from south-eastern Australia. The extent of fixed genetic differences between nematodes from different host species ranged from 0–53%. The two distinct morphological forms of the parasite found in M. rufogriseus differed at 50% of loci. Specimens present in M. fuliginosus and M. giganteus were indistinguishable genetically, as were nematodes from M. rufus and M. robustus. Of the two morphologically distinct congeners included in the analysis as controls, Rugopharynx epsilon (Johnston & Mawson, 1939) was genetically distinct (46–69% fixed genetic differences) from all specimens of the R. australis complex while R. rufogrisea Magzoub, 1964 was closely related to one of the two species occuring in M. rufogriseus. It was concluded that R. australis is a species complex, with a genetically distinct species present in M. eugenii, M. fuliginosus/M. giganteus, M. robustus/M. rufus, W. bicolor and T. billardierii, and two species in M. rufogriseus.     

Multiple phenotypes associated with Myc-induced transformation of chick embryo fibroblasts can be dissociated by a basic region mutation.

Chimaeric alleles were constructed to assay the biological functions of an N-terminal deletion and C-terminal mutations which were found in a naturally occurring mutant of feline vMyc, T17. The mutant alleles were assayed for their ability to transform chick embryo fibroblasts in vitro by a number of criteria, namely the ability to induce morphological transformation, an accelerated growth rate and growth in soft agar. Feline cMyc could transform the avian cells, whilst T17 vMyc could not, and the N-terminal deletion was responsible for conferring the primary transformation defect on the mutant protein. The C-terminal mutations which consist of a point mutation adjacent to the nuclear localisation signal and a point mutation/amino acid insertion within the basic region (BR) could, however, dissociate the Myc-induced parameters of transformation. This effect was a specific function of the BR mutation alone, and the mutation could be transferred into avian cMyc with comparable biological consequences. The BR mutation did not disrupt the sequence specific DNA binding activity of the protein in vivo, despite exerting a biological effect. These data suggest a novel phenotype where the mutation may affect a subset of Myc-regulated genes through altered DNA binding specificity or protein-protein interactions.     

Electro-immunoblotting characterization ofPseudo-nitzschia multiseries andP. pungens antigens recognized by antibodies directed against whole cells

Separate polyclonal antibodies have previously been developed against the domoic-acid-producingPseudonitzschia multiseries (=Pseudo-nitzschia pungens f.multiseries) and the non-toxicP. pungens (=P. pungens f.pungens). These antibodies bind to the surface of the diatoms as shown by immunofluorescence studies. Here we examine the molecular nature of the antigens by Western blotting (electro-immunoblotting) analysis. The major antigens for both polyclonal antibodies migrated as high molecular-weight diffuse bands, mostly remaining in the stacking gel, using an SDS-PAGE system. The antibodies prepared againstP. multiseries strongly labelled the high molecular-weight antigens of allP. multiseries strains tested and showed little reactivity towardsP. pungens extracts on Western blots.P. pungens antibodies strongly labelled high molecular-weightP. pungens antigens and faintly labelled a fewP. multiseries antigens. The selectivity of the antibodies for their respective species correlates with the results of the immunofluorescence experiments, suggesting that the antigens examined in this study are responsible for the selective labelling in immunofluorescence studies. The electrophoretic mobility and the antibody labelling of antigens were not altered by proteolytic digestion of cell pellets. However, disruption of carbohydrates in the pellets by treatment with periodic acid resulted in loss of the antigen. These data suggest that the major antigens of toxicP. multiseries and non-toxicP. pungens are high molecular-weight (°>100kDa) polysaccharides located on the surface of these diatoms.Author for correspondence     

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.     

A human glial hybrid cell line differentially expressing genes subserving oligodendrocyte and astrocyte phenotype

We have developed a series of immortal human-human hybrid cell lines that express phenotypic characteristics of primary oligodendrocytes, by fusing a 6-thioguanine–resistant mutant of the human rhabdomyosarcoma RD with adult human oligodendrocytes by a lectin-enhanced polyethylene glycol procedure. Hybrids were selected in an aminopterin-containing media. In contrast to the tumor parent cells, a hybrid clone M03.13 expressed surface immunoreactivity for galactosyl cerebroside and intracellular immunoreactivity for myelin basic protein (MBP), proteolipid protein (PLP), and glial fibrillary acidic protein (GFAP). Serum deprivation or chronic treatment with a protein kinase C activator 4-β-phorbol 12-myristate 13-acetate (PMA), but not dibutyl cyclic adenosine monophosphate induced coordinate up-regulation or de novo induction of oligodendrocyte phenotypic markers with concomitant down-regulation of GFAP expression. Consistent with immunohistochemical studies, northern blot analysis demonstrated that both MBP and PLP mRNA were up-regulated in MO3.13 cells by PMA treatment. M03.13 cells provide an immortalized clonal model system suitable for study of gene expression subserving oligodendrocyte and astrocyte phenotypes. © 1995 John Wiley & Sons, Inc.     

In vitro pluripotency of epiblasts derived from bovine blastocysts

Two experiments were conducted to compare the utility of in vitro- and in vivo-derived bovine blastocysts for the isolation of pluripotent epiblasts. In experiment 1, the inner cell masses (ICMs) of in vivo-collected blastocysts yielded a higher proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts (P = .0157). In experiment 2, ICMs of in vivo-collected blastocysts that hatched on day 8 yielded a greater proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts that hatched on day 8. The difference was reversed but smaller for blastocysts that hatched on day 9 (Interaction, P = .0125). Epiblasts from blastocysts that hatched on day 8 regardless of their source generated more differentiated cell lines in extended culture than did blastocysts that hatched on day 9. Extended epiblast culture yielded cells identifiable as products of the three embryonic germ layers that included epithelial cells, fibroblasts, neuronal cells, hepatocyte-like cells, and macrophage-like cells. Alkaline phosphatase activity combined with cell morphology identified the bovine epiblast cells and distinguished them from trophectoderm and endoderm that frequently contaminated epiblast cell cultures. In vivo-derived blastocysts, especially from early-hatching blastocysts, were a superior source of pluripotent epiblasts. Epiblast cells in this study all differentiated or senesced indicating that standard conditions for mouse embryonic stem cell culture do not maintain bovine epiblast cells in an undifferentiated state. © 1995 wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

An improved method for the isolation of type II and clara cells from mice

Identifying the causal events and temporal aspects of lung cancer development requires the ability to isolate target and nontarget cells for comparative analyses. Current methodology can either isolate only one pure specific cell population from a lung or multiple cell types at lower purity. Previous studies in our laboratory have identified the alveolar type II cell as the progenitor cell for tumor development in the A/J mouse. The purpose of this study was to develop new protocols for the isolation and culture of type II and Clara cells from the mouse lung. Both type II and Clara cells were obtained in high purity using a sequential centrifugal elutriation protocol. In the first elutriation, cell fractions were collected using a Standard chamber. The type II and Clara cell fractions were then elutriated separately (two different separations) using a Sanderson chamber. The final purity of the type II and Clara cell preparations was 73% and 76%, respectively. Colonies of 4 to 20 Clara cells exhibiting epithelial morphology were evident 1 wk after plating in low serum medium. The growth of type II cells required the addition of bronchioalveolar lavage fluid and acidic fibroblast growth factor to the medium. The isolation of viable mouse type II and Clara cells in high purity should facilitate the identification of cell-specific changes in gene expressions or in enzymatic pathways following in vivo or in vitro exposure to environmental carcinogens.     

Effects of 12 weeks of aerobic exercise plus dietary restriction on body composition, resting energy expenditure and aerobic fitness in mildly obese middle-aged women

This study investigated the effects of 12 weeks of aerobic exercise plus voluntary food restriction on the body composition, resting metabolic rate (RMR) and aerobic fitness of mildly obese middle-aged women. The subjects were randomly assigned to exercise/diet (n = 17) or control (n = 15) groups. The exercise/diet group participated in an aerobic training programme, 45–60 min · day ^–1 at 50%–60% of maximal oxygen uptake (VO_2max), 3–4 days · week^–1, and also adopted a self-regulated energy deficit relative to predicted energy requirements (–1.05 MJ · day ^–1 to –1.14 MJ · day ^–1 ). After the regimen had been followed for 12 weeks, the body mass of the subjects had decreased by an average of 4.5 kg, due mainly to fat loss, with little change of fat free mass (m _ff). The absolute RMR did not change, but the experimental group showed significant increases in the RMR per unit of body mass (10%) and the RMR per unit of m _ff (4%). The increase in RMR/m _ff was not correlated with any increase in VO_2max/m _ff. The resting heat production per unit of essential body mass increased by an average of 21%, but the resting heat production rate per unit of fat tissue mass remained unchanged. We concluded that aerobic exercise enhances the effect of moderate dietary restriction by augmenting the metabolic activity of lean tissue.