Analysis of cell-to-bubble attachment in sparged bioreactors in the presence of cell-protecting additives

To investigate the mechanisms of cell protection provided by medium additives against animal cell injury in sparged bioreactors, we have analyzed the effect of various additives on the cell-to-bubble attachment process using CHO cells in suspension. Cell-to-bubble attachment was examined using three experimental techniques: (1) cell-bubble induction time analysis (cell-to-bubble attachment times); (2) forming thin liquid films and observing the movement and location of cells in the thin films; and (3) foam flotation experiments. The induction times we measured for the various additives are as follows: no additive (50 to 500 ms), polyvinyl pyrrolidone (PVP: 20 to 500 ms), polyethylene glycol (PEG: 200 to 1000 ms), 3% serum (500 to 1000 ms), polyvinyl alcohol (PVA: 2 to 10 s), Pluronic F68 (5 to 20 s), and Methocel (20 to 60 s). In the thin film formation experiments, cells in medium with either F68, PVA, or Methocel quickly flowed out of draining thin liquid films and entered the plateau border. When using media with no additive or with serum, the flow of cells out of the thin liquid film and film drainage were slower than for media containing Pluronic F68. PVA, or Methocel. With PVP and PEG, the thin film drainage was much slower and cells remained trapped in the film. For the foam flotation experiments, a separation factor (ratio of cell concentration in the foam catch to that in the bubble column) was determined for the various additives. In the order of increasing separation factors (i.e., increasing cell attachment to bubbles), the additives are as follows: Methocel, PVA, Pluronic F68, 3% serum, serum-free medium with no additives, PEG, and PVP. Based on the results of these three different cell-to-bubble attachment experiments, we have classified the cell-protecting additives into three groups: (1) Pluronic F68, PVA, and Methocel (reduced cell-to-bubble attachment); (2) PEG and PVP (high or increased cell-to-bubble attachment); and (3) FBS (reduced cell attachment butslower drainage films compared with F68, PVA, and Methocel with some cell entrapment in those films). These phenomena are discussed in relation to the interfacial properties of the media reported in a companion Study (this issue). (c) 1995 John Wiley & Sons Inc.     

Inhibition of a Mitotic Motor Compromises the Formation of Dendrite-like Processes from Neuroblastoma Cells

Microtubules in the axon are uniformly oriented, while microtubules in the dendrite are nonuniformly oriented. We have proposed that these distinct microtubule polarity patterns may arise from a redistribution of molecular motor proteins previously used for mitosis of the developing neuroblast. To address this issue, we performed studies on neuroblastoma cells that undergo mitosis but also generate short processes during interphase. Some of these processes are similar to axons with regard to their morphology and microtubule polarity pattern, while others are similar to dendrites. Treatment with cAMP or retinoic acid inhibits cell division, with the former promoting the development of the axon-like processes and the latter promoting the development of the dendrite-like processes. During mitosis, the kinesin-related motor termed CHO1/MKLP1 is localized within the spindle midzone where it is thought to transport microtubules of opposite orientation relative to one another. During process formation, CHO1/ MKLP1 becomes concentrated within the dendrite-like processes but is excluded from the axon-like processes. The levels of CHO1/MKLP1 increase in the presence of retinoic acid but decrease in the presence of cAMP, consistent with a role for the protein in dendritic differentiation. Moreover, treatment of the cultures with antisense oligonucleotides to CHO1/MKLP1 compromises the formation of the dendrite-like processes. We speculate that a redistribution of CHO1/MKLP1 is required for the formation of dendrite-like processes, presumably by establishing their characteristic nonuniform microtubule polarity pattern.     

Phenolic compounds in seedlings of Betula pubescens and B. pendula are affected by enhanced UVB radiation and different nitrogen regimens during early ontogeny

We studied the ability of tree seedlings to respond to two environmental factors, elevated ultraviolet B (UVB) radiation and availability of nitrogen (N), at the beginning of their development. Seeds of two birch species, Betula pubescens Ehrh. (common white birch) and B. pendula Roth (silver birch), were germinated and the seedlings grown in an experimental field in eastern Finland. The experimental design consisted of a constant 50% increase in UVB radiation (including a slight increase in UVA), a UVA control (a slight increase in UVA) and a control. The seedlings were fertilized with three levels of N. The experiment lasted for 2 months; aboveground biomass was measured and the most mature leaf of each seedling was taken for the analyses of phenolics. Growth of the seedlings was not significantly affected by enhanced UVB, but was increased by increasing N. Elevated UVB induced significant changes in phenolic compounds. Quercetin glycosides were accumulated in the leaves of both species in response to UVB; this is considered to be a protective response. However, the direction of the responses of individual phenolics to different N regimens differed. In addition, concentration of soluble condensed tannins was lower at moderate N than that at lower levels of N in both species; on the contrary, in B. pubescens the concentration of insoluble condensed tannins was highest at moderate N. No significant interaction between UV and N was detected, and the responses of the two species were highly similar to UVB, while the responses to N regimens varied slightly more between species.     

Development and Evaluation of Biodegradable Chitosan Films of Metronidazole and Levofloxacin for the Management of Periodontitis

Metronidazole (MZ) and levofloxacin (LF) are widely employed for treatment of periodontitis, but high oral dose and resistance development after long-term oral administration limit their use. The aim of this study was to alleviate shortcomings in the treatment of periodontitis by fabrication of intrapocket, biodegradable films of chitosan (CS) loaded with MZ and LF meant for inserting into periodontal pockets to treat infections. The films were developed by solvent casting technique using propylene glycol as plasticizer and glutaraldehyde as crosslinking agent. Their physical characteristics, such as drug content, surface pH, swelling index, and folding endurance, exhibited results within limit. Further, FTIR and DSC studies revealed stability of films and compatibility between drugs and excipients. SEM images of films showed the presence of free drug particles on the surface causing burst effect. In vitro release in McIlvaine buffer pH 6.6 was of sustained nature assisted by the burst effect. CS and crosslinking agent concentrations negatively affected drug release and positively affected T₉₀ (time for releasing 90% of the drug) due to altered matrix density. In contrast, the plasticizer concentration increases membrane permeability and hence increased drug release, lowering T₉₀. Crosslinked films demonstrated sustained release up to 7 days. The antibacterial efficacy of films was tested on Staphylococcus aureus and Escherichia coli, indicating good antibacterial activity. Clinical trials on patients proved the therapeutic efficacy of the films by a significant (p?<?0.05) decrease in the clinical markers of periodontitis, i.e. gingival index, plaque index and pocket depth. Conclusively, the films of MZ and LF were successful tools for the management of periodontitis.     

Stimulation ofBradyrhizobium japonicum by allelochemicals from green plants

Yeast extract-mannitol broth supplemented with 3 mg.ml⁻¹ extract from aerial parts of plants was noculated withB. japonicum 3I1b110. Filter-sterilized water extracts of foxtail (Setaria viridis), lambsquarters (Chenopodium album), Johnsongrass (Sorghum halepense) and Pennsylvania smartweed (Polygonum pensylvanicum) stimulated growth of the bacterium by 3–4 times after 48 h incubation; however, similar extracts from several other plants were marginally stimulatory. Autoclaved water extracts and methanol (75%) extracts were less stimulatory than filter-sterilized water extracts. Autoclaved water extracts of nutsedge (Cyperus esculentus) and ragweed (Ambrosia psilostachya), and methanol extracts of smartweed, peat and ragweed depressed growth of the bacterium. Filter-sterilized water extracts from shoots of foxtail and lambsquarters increased growth of the bacterium linearly up to 6 mg.ml⁻¹. At that level growth in the presence of lambsquarters shoot extract was almost 20 times that of the control. Comparable growth stimulation by this extract was also found in four other strains ofB. japonicum. The water extracts from roots and stems of lambsquarters, and roots, shoots and inflorescences of foxtail at 3 mg.ml⁻¹ stimulated growth of the bacterium by more than 40 times the control.     

Lagerenyl acetate and lagerenol two tetracyclic triterpenoids with the cycloartane skeleton from Lagerstroemia lancasteri

Lagerenyl acetate and lagerenol two new tetracyclic triterpenoids with the cycloartane skeleton together with four other triterpenoids 2α-hydroxy- 3β-E-p-coumaryloxy-urs-12-en-28-oic acid (jacoumaric acid, isolated as its monoacetylmethylcarboxylate derivative), 2α-hydroxyursolic acid (isolated as its diacetate), germanicyl acetate and friedelin, and sitosterol were isolated from the leaves and twigs of Lagerstroemia lancasteri. The structures of lagerenyl acetate and lagerenol were established as 3β-acetoxycycloart-24-one and 3β-hydroxycycloart-24-one, respectively, on the basis of spectral and chemical evidence.     

Heavy metal (Pb) accumulation in metallophytes as influenced by the variations in rhizospheric and non-rhizospheric soils physico-chemical characteristics

Activities at root-soil interface determine the solubility and uptake of metals by plants. Metal accumulation in plant species (Imperata cylindrical, Cynodon dactylon, Eleucine indica, Gomphrena celosoides, Sporobolus pyramidalis, Chromolaena odorata and Rhynchospora corymbosa) growing on Pb contaminated site as influenced by variations in physico-chemical characteristics, dissolved organic matter (DOM), Pb fractionation and different functional groups (using Fourier Transmittance Infra-red) of rhizospheric and non-rhizospheric soils was assessed. The electrical conductivity (2660–5520 µs) and Pb concentrations (51390.0–64080.0 mg/kg) were more in non-rhizospheric than rhizospheric soils having 276 µs to 3160 µs EC and 3289.0 to 44850.0 mg/kg Pb. More nutrients, DOM and carbohydrates functional groups (C-O; 1100 -1000 and O-H; 3700–3600) were found in rhizospheric compared to non-rhizospheric soils. The pH was slightly acidic (5.0–5.54) and E. indica with the lowest pH (5.0) accumulated highest Pb concentrations in shoot (8030 mg/kg) and root (16380 mg/kg) while C. odorata with highest values of pH, P, Ca and Mg in rhizospheric soil accumulated the least (root; 331.6 and shoot: 209.0 mg/kg). Pb was more in organic and residual fractions of rhizospheric and non-rhizospheric soils respectively. Reduction in pH, EC coupled with nutrients and DOM availability increased Pb uptake by plants.     

Isotherm and kinetic models and cell surface analysis for determination of the mechanism of metal sorption by Aspergillus versicolor

The isolate Aspergillus versicolor was obtained from an estuary, which is exposed to metal contamination. It was found to have a good metal tolerance and sorption capacity. Further studies revealed that the rate of metal removal from solution is very rapid in the first 5-10 min, and is favoured by a pH of 6.0. The biosorption data obtained was explained by the Freundlich adsorption isotherm model and followed a pseudo-second order kinetics reaction. The fungus showed a higher accumulation of fatty acids when grown in presence of metals as compared to the mycelium grown in absence of the metal; there was also an increase in the saturation index of fatty acids in presence of Cu(2+) which serves as a protective mechanism for the fungus. Fourier Transform Infrared, scanning electron microscopy and EDAX analysis indicated that metal removal from solution by A. versicolor occurred by a passive adsorption to the fungal cell surface, involving an ion exchange mechanism.