The effects of Cu on the adenylate energy charge of open ocean phytoplankton

The effects of short-term, acute Cu exposure (6 h) on the adenylateenergy charge (EC_A) of open-ocean phytoplankton populations(northeastern equatorial Pacific) were investigated. Energycharge remained at {small tilde}0.77 over the range of Cu additions(0.025 – 5.µg l^–1), even though ¹⁴C uptakeand total adenylate levels (ATP + ADP + AMP) were reduced byas much as 60%. These findings suggest that EC_A alone is nota sensitive indicator of acute sublethal metal effects on phytoplankton. ¹This research was supported by the NSF Biological OceanographyProgram grant #OCE 81-17286.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

An allied reprogramming,selection, expansion and differentiation platform for creating hiPSC on microcarriers

ObjectivesInduced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.Materials and MethodsFive sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.ResultsImprovement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50‐fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β‐catenin, E‐cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ‐layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.ConclusionsOur method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

We have developed an allied protocol for reprogramming, selecting, expanding and differentiating human pluripotent stem cells on Microcarriers (designated as RepMC). This method allows faster reprogramming, selecting 30‐50‐fold more candidates for characterization and also allows us to find high quality candidates that differentiate to cardiomyocytes and blood lineages. Mechanistically, this method appears to accelerate the induction, maturation and stabilization phases of reprogramming. Our findings help simplify the process of deriving and expanding iPSCs for therapeutic applications, offering a robust and scalable suspension platform for large‐scale generation of clinical grade iPSCs.     

Staining of Some Specific Regions of Human Chromosomes,particularly the Secondary Constriction of No. 9

SEVERAL procedures have been described recently which produce specific patterns of differential staining in human chromosomes^1–9. Techniques which involve DNA denaturation and reannealing reveal deeply stained areas on centromere and secondary constriction regions which have been equated with constitutive heterochromatin⁹.     

The mode of action of thidiazuron: auxins,indoleamines, and ion channels in the regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.     

Evolution of resident bird breeding phenology in a landscape with heterogeneous resource phenology and carryover effects

It is generally expected that, in environments with pronounced seasonal resource peaks, birds’ reproductive success will be maximised when nestlings’ peak food demand coincides with the timing of high food availability. However in certain birds that stay resident over winter, earlier breeding leads juveniles to join the winter flock earlier, which by the prior residence effect increases their success in breeding territory competition. This trade-off between reproduction and competition may explain why, in certain species, breeding phenology is earlier and asynchronous with the resource. This study extends a previous model of the evolution of breeding phenology in a single habitat type to a landscape with two habitat types: ‘early’ and ‘late’ resource phenology. The offspring’s natal habitat type has a carryover effect upon their competitive ability regardless of which habitat type they settle in to potentially breed. We find that, when the difference in resource phenology between habitats is small (weak carryover effect), breeding phenology in the late habitat evolves to occur earlier and more asynchronously than in the early habitat, to compensate for the competitive disadvantage to juveniles raised there. However if the difference is large (strong carryover effect), then the reproductive cost of earlier breeding outweighs the benefit of the compensation, so instead breeding phenology in the late habitat evolves to become more synchronous with the resource. Recruitment is generally asymmetric, from early to late habitat type. However if the early habitat is less frequent in the landscape or produces fewer offspring, then the asymmetry is reduced, and if there is some natal habitat-type fidelity, then recruitment can have an insular pattern, i.e. most recruits to each habitat type come from that same habitat type. We detail the different scenarios in which the different recruitment patterns are predicted, and we propose that they have implications for local adaptation.     

Auxin-like activity of extract from hypertrophied tissue of <Emphasis Type="Italic">Acacia eburnea</Emphasis> infected with <Emphasis Type="Italic">Ravenelia esculenta</Emphasis>

Ravenelia esculenta Naras. and Thirum. is a rust, pathogenic to Acacia eburnea Willd. The infection leads to hypertrophy changing the morphology with bizarre shapes of plant organs. Healthy and infected tissues were subjected to extraction of IAA and indole derivatives and were estimated by spectrophotometric methods. The hypertrophy produced was presumed to be due to increase in the indole-3-acetic acid (IAA) content in the infected tissue, however, the amount of IAA in infected tissues decreased with the progression of disease. Concomitantly, the infected tissue showed the presence of a novel, slow migrating, indole derivative on TLC. Cultured shoot tips of Withania somnifera were dosed with the methanolic extract of the infected hypertrophied tissue (MEHT) (0.25, 0.5, 0.75, 1.00 and 1.25 mg/l). The stimulation in shoot growth along with profuse rooting was observed in a dose dependent manner with maximum at 1.00 and 1.25 mg/l concentration.