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A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM8P medium with 0.45 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 µM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 µM indole-3-butyric acid (IBA) and 0.93 µM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient β-glucuronidase (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and 100 μF in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1–2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.

A method for plant regeneration in Robinia pseudoacacia L. from cell suspension culture was established. Non regenerative friable callus from hypocotyls and cotyledon explants from in vitro raised seedling induced on solid Murashige and Skoog (MS) medium supplemented with 0.05 mg dm−3 2,4-dichlorophenoxyacetic acid (2,4-D) was used for initiation of cell suspension cultures on same MS medium but without agar. Single cells were isolated after 3 d and the optimum cell density was 1–3 × 104 cells per cm3 of the liquid MS medium. Plating efficiency was 29.6 % and callus formed within 4 weeks was subcultured and transferred to solid MS medium supplemented with 0.6 mg dm−3 benzyladenine (BA) along with 0.05 mg dm−3 α-naphthalene-1-acetic acid (NAA) for the induction of adventitious bud primordia. The shoots developed were isolated and re-cultured on MS medium containing 0.6 mg dm−3 BA. These microshoots after dipping in 1–2 cm3 of 10 mg dm−3 indole-3-butyric acid (IBA) for 24 h in dark were cultured on half strength solid MS medium supplemented with 0.05 % charcoal and showed 80–82 % rooting within 4 weeks.

Electroless deposition of nickel and cobalt from alkaline baths as well as their codeposition with SiC particles were compared. It was found that despite similarity in properties of the metals their behavior in the electroless process was different. Maleic acid was used to stabilize plating baths, however the optimal concentration of the additive depended on the type of deposited metal. The electroless deposition characterized with low plating efficiency and it was higher for nickel than for cobalt. The composition as well as the morphology of Ni–P and Co–P deposits were also dissimilar. SiC particles inhibited the metal deposition, affected the plating potential, but did not alter the matrix composition. SiC entrapment was more preferential within the cobalt matrix than the nickel one. Some adsorption phenomena on SiC powder were also studied.

The electrodeposition of nickel–iron alloys has been studied with different conditions of pH, substrate and magnetic field induction. All experimental conditions influence the current efficiency value and the alloy composition. For low current densities, alloys obtained at pH 3 present a smaller nickel percentage onto tin substrate compared to copper substrate. In the same time, the plating efficiency is improved onto tin electrode. On the other hand, if electrolyze is realized with magnetic field superimposition, the nickel percentage and the current efficiency are together reduced. For constant conditions of pH, substrate and magnetic induction, we observe that more the efficiency current is low, more the alloy composition is rich in nickel. All these effects relating to the plating efficiency and to the Ni–Fe alloy composition are elucidated using the hydrogen evolution reaction (HER).

Exposure of Agrobacterium tumefaciens to menadione, cumene hydroperoxide, and diamide strongly induced trxA expression. The trxA mutant showed a reduction in the aerobic growth rate and plating efficiency and was cytochrome c oxidase negative. Atypically, the mutant has decreased resistance to menadione but an increased H 2 O 2 resistance phenotype.

Protoplasts isolated from wild cotton Gossypium davidsonii were cultured in KM8P medium supplemented with different phytohormones. The most effective combination was 0.45 µM 2,4-dichlorophenoxyacetic acid, 2.68 µM α-naphthaleneacetic acid and 0.93 µM kinetin and the division percentage at the 8th day was 30.78 ± 3.04 %. The density of protoplasts at 2–10 × 105 cm−3 was suitable for protoplast division and calli formation, with a division percentage of 32.21 ± 3.64 % and a plating efficiency of 9.12 ± 2.61 % at the 40th day. The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol. Protoplasts were cultured in three ways, a double-layer culture system, with liquid over solid medium was proved to be the best way. Embryo induction was further increased by addition of 0.14 µM gibberellic acid.

Sunflower, as one of the most important oil-producing crops, represents an important target for genetic improvement through gene transfer or somatic hybridization. Unfortunately, sunflower is recognized as recalcitrant to in vitro culture. The aim of our paper was to improve sunflower protoplast regeneration. Three cultivars (Romanian hybrids) and one inbred line were used for protoplast isolation from etiolated hypocotyls. Isolated protoplasts were embedded in alginate disks and cultured in two plating densities, using two culture regimes as indicated by previous authors. Plating efficiency, callus development and plant regeneration were evaluated as well as old callus histology. In cv. ‘Select’, the effects of 1:50 haemoglobin and 1 mM spermidine were assayed on asymmetric division and/or plating efficiency. Plant regeneration from hypocotyl protoplasts was achieved for two cvs., ‘Florom 328’ and ‘Turbo’, with the former proving once more its totipotency. The best culture regime proved to be as recommended by Krasnyanski and Menczel (1993), but the best density in the culture medium was the highest ever tested, 8 × 105 pp ml−1. Moreover, the histology of old green compact protoplast-derived callus revealed a very well organized structure suggesting senescence. In the non-responsive cv. ‘Select’, haemoglobin was found to stimulate protoplast asymmetric division and the development of heart-shaped embryo-like structures, while spermidine stimulated overall protoplast plating efficiency.

Objective To optimize transduction efficiency of mobilized CD34 + cells with serotype-5 adenoviruses (Ad5s), we investigated the activity of the chemical cocktail BoosterExpress Reagent in enhancing Ad5-mediated gene transfer into CD34 + cells. Methods Enriched CD34 + cells were transduced with three different Ad5s at increasing multiplicity of infections (MOIs) in the presence and absence of BoosterExpress Reagent. Percentages of transduced cells and levels of transgene expression were quantified by flow cytometry. Propidium iodide staining and colony growth were used to assess the toxicity of the transduction protocol. Expression of adenovirus receptors was investigated by flow cytometry. Results Irrespective of the Ad5 used, transduction with BoosterExpress Reagent using an MOI of 500 resulted in an average six- to seven-fold increase of transduction efficiencies and 1.5- to 2-fold increase of the levels of transgene expression, which could be detected up to 7 days post-transduction. Although BoosterExpress Reagent alone did not affect the plating efficiency of CD34 + cells, adenovector transduction plus BoosterExpress Reagent significantly reduced the plating efficiency due to the marked increase of transduced cells. However, adenoviral transduction in the presence of BoosterExpress Reagent failed to significantly reduce the recovery of CD34 + cells as compared with transduction in the absence of the compound. Coxsackievirus and adenovirus receptor as well as α v β 3 , α v β 5 , α 5 , and β 1 integrins were upregulated by BoosterExpress Reagent. Conclusions BoosterExpress Reagent allows high-levels of durable transgene expression, thus making CD34 + cells a suitable target for Ad5-mediated gene transfer.

Somatic hybridization offers the possibility of manipulating plant genomes between species. Successful application of this technique in plant cultivar improvement requires a high efficient plant regeneration system from protoplasts. A method for preparing viable protoplasts from somatic embryogenic cell suspension cultures was established in Vitis vinifera grape 'Autumn Royal Seedless' and V. rotundifolia 'Tara'. Somatic hybridization was attempted between protoplast derived from embryogenic cell lines and leaf protoplasts isolated from in vitro-grown plants 'Orlando Seedless', 'Cabernet Sauvignon', and 'Alachua' by means of polyethylene glycol (PEG). When cultured on BH3 and modified MS basal media containing 20 g/L maltose and 0.5 g/L glutamine supplemented with 5.0 μM β-naphthoxyacetic acid (NOA), the fusion products exhibited cell wall formation, cell division, and callus formation about 4 days after the cultivation. The highest plating efficiency was obtained on MS basal medium.

A semiselective medium was developed for isolating Xanthomonas campestris pv. musacearum (Xcm) from infected banana plants, soil and insect vectors. The new medium was named cellobiose-cephalexin agar (CCA) and it contained (L−1): 1 g yeast extract, 1 g glucose, 1 g peptone, 1 g NH4Cl, 1 g MgSO4·7H2O, 3 g K2HPO4, 1 g beef extract, 10 g cellobiose, 14 g agar, 40 mg cephalexin, 10 mg 5-fluorouracil and 120 mg cycloheximide. The medium was evaluated for selectivity using 21 bacterial isolates and for plating efficiency using Xcm. The bacterial isolates included a soilborne Xanthomonas species and three pathogenic Xanthomonas strains that infect cassava, cabbage and beans. Although the plating efficiency of Xcm on CCA was lower (59%) than on non-selective yeast extract peptone glucose agar (YPGA), its selectivity was significantly higher, averaging 60 and 82%, when isolating from banana fruits and soil, respectively. CCA was also superior when isolating Xcm from insect vectors, with selectivity of 48–75%, compared with 8–17% on YPGA. Xanthomonas campestris pv. phaseoli did not grow on CCA, while X. campestris pv. campestris and X. axonopodis pv. manihotis grew, but their colonies were smaller than those of Xcm. Twenty-nine out of 33 suspected Xcm strains isolated from plants, soil and insects using CCA were pathogenic when inoculated onto banana plants, indicating that CCA can be a reliable tool in isolating Xcm populations. The medium should prove useful in studies on ecology, epidemiology and management of the banana bacterial wilt pathogen that is currently ravaging bananas in East and Central Africa.

Object An understanding of single glioma cell invasion has been limited by the static picture provided by histological studies. The ability to nondestructively assess cell invasion dynamically in a full 3D volume would improve the quality and quantity of information available from both in vivo and in vitro experiments. The purpose of this study was to observe glioma cell invasion in a 3D in vitro model using a microimaging protocol at 1.5 tesla and to assess the uptake of micron-sized particles of iron oxide (MPIO) and the consequent effects on cell function. Methods Rat C6 glioma cells were labeled with MPIO to a sufficient extent to allow single cell detection in vitro without significant effects on cell proliferation or plating efficiency. When placed on agar-coated plates, the cells formed stable multicellular tumor spheroids (MCTSs), which were embedded in collagen type I gel and serially visualized using magnetic resonance (MR) imaging and phase-contrast microscopy over 8 days. The MCTSs initially appeared as large susceptibility artifacts on MR images, but within 2 days, as cells moved away from the main MCTS, small discrete areas of signal loss, possibly due to single cells, could be observed and tracked. Conclusions Glioma cell invasion can be nondestructively observed using MR imaging. The sensitivity of MR imaging, along with its ability to represent full 3D volumes noninvasively over time, makes it ideal for longitudinal in vivo cell tracking studies.

Acrolein is a highly reactive alpha,beta-unsaturated aldehyde produced endogenously during lipid peroxidation and naturally distributed pervasively in living environments, posing serious threats to human health if not properly metabolized. In this study, we report aldose reductase-like-1 (ARL-1) as a novel enzyme that catalyzes the reduction of acrolein and protects cells from their toxicity. Using purified ARL-1 protein, we determined its enzymatic activity in response to acrolein and defined its steady-state kinetics with K(m) and V(max) at 0.110 +/- 0.012 mM and 3122.0 +/- 64.7 nmol/mg protein/min, respectively. By introducing a functional Enhanced Green Fluorescent Protein (EGFP)/ARL-1 fusion protein into 293T cells, we demonstrated that plating efficiency in liquid culture and focus formation in soft agar increased by more than 60% (p0.05), compared to the vector control cells. More significantly, at a low dose of 5 microM acrolein, EGFP/ARL-1 expression enhanced both plating efficiency and focus formation by more than threefold, and the foci (in soft agar) of 293T cells expressing EGFP/ARL-1 were significantly larger than those of the vector control cells. At high concentrations of acrolein (25 and 50 microM), EGFP/ARL-1 protein prevented oncotic death of 293T cells induced by acrolein. In summary, our data demonstrated for the first time that the ARL-1 protein protects 293T cells from acrolein toxicity. Due to the high toxicity and wide distribution of acrolein, this finding is important to the understanding of its detoxification mechanisms.

Our experimental approach toward the development of new islet-based treatment for diabetes mellitus has been the creation of a monolayered islet cell construct (islet cell sheet), followed by its transplantation into a subcutaneous pocket. Previous studies describe rat laminin-5 (chain composition: a3, b3, g2) as a suitable extracellular matrix (ECM) for surfaces comprised of a coated temperature-responsive polymer, poly( N-isopropylacrylamide) (PIPAAm). To progress toward the clinical application of this approach, the present study attempted to identify an optimal human ECM as a coating material on PIPAAm surfaces, which allowed islet cells to attach on the surfaces and subsequently to be harvested as a monolithic cell sheet. Dispersed rat islet cells were seeded onto PIPAAm dishes coated with various human laminin isotypes: human laminin (HL)-211, HL-332, HL-411, HL-511, and HL-placenta. Plating efficiency at day 1, the confluency at day 3, and glucose-stimulated insulin secretion test at day 3 were performed. The highest value of plating efficiency was found in the HL-332-PIPAAm group (83.1 ± 0.7%). The HL-332-PIPAAm group also showed the highest cellular confluency (98.6 ± 0.5%). Islet cells cultured on the HL-332-PIPAAm surfaces showed a positive response in the glucose-stimulated insulin secretion test. By reducing culture temperature from 37°C to 20°C in the HL-332-PIPAAm group, cells were able to be harvested as a monolithic islet sheet. The present study showed that HL-332 was an optimal human-derived ECM on a PIPAAm coating for preparing islet cell sheets.

Neonatal and adult keratinocytes isolated from thin sections of split-thickness skin by trypsin-release show a preferential and strong attachment to collagen when compared to plastic, fibronectin-coated plastic, glass, or agar gels. We have investigated the reactive groups of keratinocytes and collagen required for this interaction and have determined the kinetics of attachment. At 37 degrees C both neonatal and adult keratinocytes show a rapid and irreversible attachment to collagen, reaching a plateau phase at 30-60 minutes. The cells cannot be replaced from the gel by extensive washing or by conditions normally expected to break ionic bonds. Chilling to 0 degree C before plating completely inhibits attachment, and heating at 37 degrees C reverses the inhibition. One cycle of freezing and thawing of cells inhibits the interaction. Removal of sialic residues from keratinocytes before plating with neuraminidase, or oxidation of sugars with periodate, does not inhibit attachment or growth, indicating that cell carbohydrates are not required for interaction with collagen. Neither denaturation of collagen with 8 M urea nor oxidation of sugar side chains on the gel with periodic acid affects attachment or growth. However, reaction of free-SH groups with iodoacetic acid or -NH2 groups with dinitrofluorobenzene of the gel completely inhibits growth. Blocking the guanidyl residues of collagen arginine with cyclohexanedione markedly alters all aspects of attachment, growth, and morphology, producing new and completely unique growth patterns. These studies indicate that specific chemical groups on collagen affect keratinocyte-matrix interactions and that the availability of specific residues in collagen directly influences growth and maturation. Most vertebrate cells remain closely associated with extracellular collagenous substances throughout their lifespan. The collagen may be present in both collagen fibers and in reticular fibers as well as in basement membranes. The way cells interact with and are anchored to these various substrata influences a number of important cellular functions including growth and maturation and the synthesis of extracellular matrix components. Skin epithelial cells display a particularly striking and strong dependence on collagen for growth. When plated on a collagen gel, the plating efficiency and growth is increased several-fold compared to other substrates such as glass, plastic, or agar. More recently, the initial observations on the selective attachment of keratinocytes to collagen gels have been extended by Murray et al., who demonstrated that guinea pig keratinocytes show increased plating efficiencies on Type IV collagen gels. In these studies, we have examined the mechanisms for keratinocyte-collagen interaction, and described the kinetics of attachment, the reactive sites on the cell and collagen, and the effects of chemical modification of collagen on the expression of the keratinocytes phenotype.

Purpose To investigate the radiosensitivity of primary glioma stem cell (GSC) cultures with different CD133 status in a 3-dimensional (3D) model after photon versus proton versus carbon irradiation. Methods and Materials Human primary GSC spheroid cultures were established from tumor specimens of six consented glioblastoma patients. Human U87MG was used as a classical glioblastoma radioresistant cell line. Cell suspensions were generated by mechanical dissociation of GSC spheroids and embedded in a semi-solid 3D matrix before irradiation. Spheroid-like colonies were manually counted by microscopy. Cells were also recovered and quantified by fluorescence. CD133 expression and DNA damage were evaluated by flow cytometry. Results The fraction of CD133 + cells varied between 0.014% and 96% in the six GSC cultures and showed a nonsignificant correlation with plating efficiency and survival fractions. The 4 most photon-radioresistant GSC cultures were NCH644, NCH421k, NCH441, and NCH636. Clonogenic survival for proton irradiation revealed relative biologic effectiveness (RBE) in the range of 0.7-1.20. However, carbon irradiation rendered the photon-resistant GSC cultures sensitive, with average RBE of 1.87-3.44. This effect was partly attributed to impaired capability of GSC to repair carbon ion–induced DNA double-strand breaks as determined by residual DNA repair foci. Interestingly, radiosensitivity of U87 cells was comparable to GSC cultures using clonogenic survival as the standard readout. Conclusions Carbon irradiation is effective in GSC eradication with similar RBE ranges approximately 2-3 as compared with non-stem GSC cultures (U87). Our data strongly suggest further exploration of GSC using classic radiobiology endpoints such as the here-used 3D clonogenic survival assay and integration of additional GSC-specific markers.

Radiation-induced bystander effects (RIBEs) are detected in cells that are not irradiated but receive signals from treated cells. The present study explored these bystander effects in a U87MG multicellular tumour spheroid model. A medium transfer technique was employed to induce the bystander effect, and colony formation assay was used to evaluate the effect. Relative changes in expression of BAX, BCL2, JNK and ERK genes were analysed using RT-PCR to investigate the RIBE mechanism. A significant decrease in plating efficiency was observed for both bystander and irradiated cells. The survival fraction was calculated for bystander cells to be 69.48% and for irradiated cells to be 34.68%. There was no change in pro-apoptotic BAX relative expression, but anti-apoptotic BCL2 showed downregulation in both irradiated and bystander cells. Pro-apoptotic JNK in bystander samples and ERK in irradiated samples were upregulated. The clonogenic survival data suggests that there was a classic RIBE in U87MG spheroids exposed to 4 Gy of X-rays, using a medium transfer technique. Changes in the expression of pro- and anti-apoptotic genes indicate involvement of both intrinsic apoptotic and MAPK pathways in inducing these effects.

Obiective: To study the change of p53 expression in BEAS-2B cells malignant transformation process, and detect the role of p53 expression for lung cancer early diagnosis. Method: BEAS-2B cells acute expose NNK at 500 μg/ml for 24 hours, and these cells (BEAS-2BNNK cells) were subcultured continuously in vitro, Biological characteristics and ultrastructure of them were studied with Colony formation assay and electron microscopy. The change p53 expression of BEAS-2B cells were detected by immunohistochemical method. Results: The serum resistance was appeared in the 5th passage of BEAS-2BNHK cells and these cells could not grow into tumor in nude micei¼›The plating efficiency of the 15th passage of BEAS-2BNNK cells in soft agar (0.032%) increased 13.9 fold Compared with that of control group cells(0.0023%)P

The relative sensitivities of different tumor cells to photodynamic therapy (PDT) with benzoporphyrin derivative monoacid ring A (BPD-MA) were compared in the four tumor cells. A good correlation was observed between the cell survival at 0.1 μg/ml of BPD-MA and sensitizer uptake/10 6 cells ( r = −0.99) or the plating efficiency of cells ( r = 0.99). At 3 h after the irradiation, a significant difference was observed in the proportion of apoptotic cells among the four tumor cells ( p = 0.024). In conclusion, cell responses to PDT depend on the several factors such as the cell line, photosensitizer dose, and fluence.

We found recently that, in yeast cells, the heterologous expression of Bax induces a loss of plating efficiency different from that induced by acute stress because it is associated with the maintenance of plasma membrane integrity (Camougrand, N., Grelaud-Coq, A., Marza, E., Priault, M., Bessoule, J. J., and Manon, S. (2003) Mol. Microbiol. 47, 495-506). Bax effects were neither dependent on the presence of the yeast metacaspase Yca1p and the apoptosis-inducing factor homolog nor associated with the appearance of typical apoptotic markers such as metacaspase activation, annexin V binding, and DNA cleavage. Yeast cells expressing Bax instead displayed autophagic features, including increased accumulation of Atg8p, activation of vacuolar alkaline phosphatase, and the presence of autophagosomes and autophagic bodies. However, the inactivation of autophagy did not prevent and actually slightly accelerated Bax-induced loss of plating efficiency. On the other hand, Bax expression induced a fragmentation of the mitochondrial network, which retained, however, some level of organization in wild-type cells. However, when expressed in cells inactivated for the gene UTH1, previously shown to be involved in mitophagy, Bax induced a complete disorganization of the mitochondrial network. Interestingly, although mitochondrially targeted green fluorescent protein was slowly degraded in the wild-type strain, it remained unaffected in the mutant. Furthermore, the slow loss of plating efficiency in the mutant strain correlated with a loss of plasma membrane integrity. These data suggest that Bax-induced loss of growth capacity is associated with maintenance of plasma membrane integrity dependent on UTH1, suggesting that selective degradation of altered mitochondria is required for a regulated loss of growth capacity.

We describe here an efficient and reproducible protocol for isolation and culture of protoplasts from Ulmus minor. Different sources of donor tissues were tested for protoplast isolation: callus and juvenile leaves from in vitro and greenhouse plants. Several combinations and concentrations of hydrolytic enzymes were used. Comparative tests between Cellulase Onozuka R10 and Cellulase Onozuka RS were made and the last one proved to be more efficient. Both the pectinases used, Macerozyme Onozuka R10 and Pectinase (Sigma®), were efficient in protoplast isolation and there was no need for a more active pectinase. In vitro leaves proved to be the best source for protoplast isolation and produced an average of 3.96 × 107 protoplasts per gram of fresh weigh. Elm mesophyll protoplasts were cultured using the advantageous method of agarose droplets and a modification of the Kao and Michayluk culture medium, using two plating densities (1 × 105 and 2 × 105 protoplasts ml−1). Protoplast division and evolution into colonies and microcalli was promoted in the agarose droplets plated at 2 × 105 protoplasts ml−1. Ten weeks after protoplast culture initiation a plating efficiency of 2.7% was attained and the bigger microcalli, with at least 0.5 mm diameter, were transferred to a solid medium previously used for the production of embryogenic callus.

This lab reported previously that the plating efficiency of a herpes simplex virus type 1 ICP0-null mutant was enhanced upon release from an isoleucine block which synchronizes cells to G 1 phase (W. Cai and P. A. Schaffer, J. Virol. 65:4078-4090, 1991). Peak plating efficiency occurred as cells cycled out of G 1 and into S phase, suggesting that the enhanced plating efficiency was due to cellular activities present in late G 1 /early S phase. We have found, however, that the enhanced plating efficiency did not occur when cells were synchronized by alternative methods. We now report that the plating efficiency of ICP0 − viruses is not enhanced at a particular stage of the cell cycle but rather is enhanced by specific cellular stresses. Both the plating and replication efficiencies of ICP0 − viruses were enhanced as much as 25-fold to levels similar to that of wild-type virus when monolayers were heat shocked prior to infection. In addition to heat shock, UV-C irradiation but not cold shock of monolayers prior to infection resulted in enhanced plating efficiency. We further report that the effect of cellular stress is transient and that cell density rather than age of the monolayers is the primary determinant of ICP0 − virus plating efficiency. As both cell stress and ICP0 are required for efficient reactivation from latency, the identification of cellular activities that complement ICP0 − viruses may lead to the identification of cellular activities that are important for reactivation from neuronal latency.

The improved performance and electrodeposition behaviour of platinum baths containing [Pt(NH3)3(NO2)](NO2) and [Pt(NH3)4](X) (X = 2Cl−, 2NO3− and 2NO2−) complex species in phosphate buffer onto titanium is described. The cathode efficiency for each bath has been determined and compared. Structures of the deposited coatings were evaluated and discussed with the aid of SEM and XRD. The role of counter anions (X) coupled with pH was investigated. The study showed that electrolytes with X = NO2− exhibited higher plating efficiency in the pH range 10·4–10·6 containing phosphate buffer, demonstrating the selectivity for specific counter anions and pH range. The current efficiency of this system is much higher than that of the bath based on Pt–P salt.

Aims: To elucidate the factors influencing the sensitivity of Bacillus subtilis spores in killing and disrupting by mechanical abrasion, and the mechanism of stimulation of spore germination by abrasion. Methods and Results: Spores of B. subtilis strains were abraded by shaking with glass beads in liquid or the dry state, and spore killing, disruption and germination were determined. Dormant spores were more resistant to killing and disruption by abrasion than were growing cells or germinated spores. However, dormant spores of the wild-type strain with or without most coat proteins removed, spores of strains with mutations causing spore coat defects, spores lacking their large depot of dipicolinic acid (DPA) and spores with defects in the germination process exhibited essentially identical rates of killing and disruption by abrasion. When spores lacking all nutrient germinant receptors were enumerated by plating directly on nutrient medium, abrasion increased the plating efficiency of these spores before killing them. Spores lacking all nutrient receptors and either of the two redundant cortex-lytic enzymes behaved similarly in this regard, but the plating efficiency of spores lacking both cortex-lytic enzymes was not stimulated by abrasion. Conclusions: Dormant spores are more resistant to killing and disruption by abrasion than are growing cells or germinated spores, and neither the complete coats nor DPA are important in spore resistance to such treatments. Germination is not essential for spore killing by abrasion, although abrasion can trigger spore germination by activation of either of the spore's cortex-lytic enzymes. Significance and Impact of the Study: This work provides new insight into the mechanisms of the killing, disruption and germination of spores by abrasion and makes the surprising finding that at least much of the spore coat is not important in spore resistance to abrasion.

An efficient approach was described for the culture of protoplasts isolated from chrysanthemum, and factors affecting the plating efficiency, defined as the number of protoplasts developing microcolonies divided by the number of cultured protoplasts, were investigated here. A yield of 4.86 ± 0.22 × 105 mesophyll protoplasts per gram fresh weight were achieved by an enzymolysis procedure. The viability of fresh isolated protoplasts was 64.5 ± 8.2% as confirmed by Evans blue staining. Sustained cell division and microcolony formation from the protoplasts were supported by modified Murashige and Skoog medium, complemented with 1:4 diluted conditioned medium harvested from three different callus suspension cultures. Results showed that the plating efficiency was obviously influenced by various concentrations of agarose and antibiotic, and significantly increased by conditioned medium harvested from chrysanthemum suspensions, while the other two species had a positive effect generally. Results also indicated that the growth-stimulative effect was reduced when the conditioned medium was stored for 6–14 days prior to use, suggesting the unstable characteristic of conditioning factors.

An efficient and reproducible protocol is described for the regeneration of Cinnamomum camphora protoplasts isolated from cultured embryogenic suspension cells. Maximum protoplast yield (13.1+/-2.1x10(6)/g FW) and viability (91.8+/-3.8%) were achieved using a mixture of 3% (w/v) cellulase Onozuka R10 and 3% (w/v) macerozyme Onozuka R10 in 12.7% (w/v) mannitol solution containing 0.12% (w/v) MES, 0.36% (w/v) CaCl(2).2H(2)O, and 0.011% (w/v) NaH(2)PO(4).2H(2)O. First divisions occurred 7-10 days following culture initiation. The highest division frequency (24.6+/-2.9%) and plating efficiency (6.88+/-0.8%) were obtained in liquid medium (MS) supplemented with 30 g l(-1) sucrose, 0.7M glucose, 0.1 mg l(-1) NAA, 1.0 mg l(-1) BA, and 1.0 mg l(-1) GA(3). After somatic embryo induction and then shoot induction, the protoplast-derived embryos produced plantlets at an efficiency of 17.5%. Somatic embryos developed into well-rooted plants on MS medium supplemented with 1.0 mg l(-1) 3-indole butyric acid (IBA). Regenerated plants that transferred to soil have normal morphology.

The methanogenic archaeon Methanosarcina mazei strain Gö1 has so far proven to be genetically intractable due to its low plating efficiency on solid medium and the lack of an effective transformation method. Here, we report the first significant improvement in plating efficiency (up to 10%), which was achieved by (1) selecting for a spontaneous mutant of M. mazei that shows significantly higher resistance to mechanical stress during spreading an agar plates, and (2) plating the cells in 0.5% top agar with trimethylamine as a carbon and energy source under a H2S-containing atmosphere (0.1%). Using this mutant we succeeded in establishing a liposome-mediated transformation protocol, which for the first time allowed genetic manipulation of the M. mazei Gö1 strain. We further report on the construction of the first chromosomal deletion mutant of M. mazei by means of homologous recombination. Characterization of this mutant strain revealed that M. mazei cells lacking a functional glnK1-gene exhibited a partial growth defect under nitrogen limitation when molecular nitrogen was used as the sole nitrogen source. Quantitative RT-PCR analysis, however, showed that genes involved in nitrogen assimilation or nitrogen fixation are transcribed in the glnK1 mutant as in the wild type. Thus, we propose that the archaeal GlnK1 protein is not directly involved in the transcriptional regulation of genes involved in nitrogen metabolism, but rather affects their protein products directly.

The sol–gel method was employed to synthesize hydroxyapatite (HAp) coatings modified with Ag or Zn ions onto Ti-6Al-4V substrate. A bacterial strain Streptococcus mutans (S. mutans) and a human gingival fibroblast (HGF-1) cell line were used to investigate the antimicrobial effect and biocompatibility, respectively. HAp coatings containing 100 ppm Ag+ ions suppressed the growth of S. mutans. An apparent inhibition zone around the HAp coating was further observed at Ag+ concentration up to 10,000 ppm. However, for coatings containing Zn2+ ions, a clear inhibition zone was observed at Zn2+ concentration of 10,000 ppm. Nevertheless, the results of HGF-1 cultivation demonstrated that the Zn2+-modified HAp coatings exhibited better attachment and spread of HGF-1 than did the Ag+-modified coatings. Zn2+ modified HAp coatings also increased the plating efficiency of HGF-1 cells. The cytotoxicity associated with the addition of Ag and the cell-conductive capacity associated with the addition of Zn are proportional to the added concentration, from 100 to 10,000 ppm. The dosages of both Ag+ and Zn2+ ions that should be added to HAp coatings were considered to prevent infection and improve biocompatibility. The results of this study ensure that HAp coatings modified with a moderate amount of Ag/Zn efficiently resist microorganisms and improve biocompatibility. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006

Although epidemiological findings suggest that normal humans are resistant to Paracoccidioides brasiliensis infection, the host defense mechanisms against this fungus have not been fully understood. Here we examined human leukocytes for antifungal activity against yeast cells of this fungus, using an improved mycological culture medium with high plating efficiency for the yeast cell. In an attempt to minimize the impairment of leukocyte activities during the isolation process, leukocytes removed by centrifugation from a buffy coat of peripheral blood were used in the antifungal assay without further fractionation. The leukocytes thus prepared effectively killed P. brasiliensis yeast cells within the first 4 h of co-culture. Adding interferon-gamma (37 ng/ml), granulocyte-macrophage colony-stimulating factor (5 ng/ml), interleukin (IL)-1beta (12 ng/ml), or IL-4 (12 ng/ml) to the assay system enhanced the leukocyte antifungal (growth inhibitory) activity by 48 h. By contrast, addition of IL-8 (50 ng/ml) impaired the leukocyte activity. Tumor-necrosis factor-alpha (50 ng/ml) or IL-10 (25 ng/ml) had no effect in this respect. Dexamethasone (1 micromol/l) reduced the antifungal activity of leukocytes. This is the first demonstration that human leukocytes are able to effectively kill yeast cells of P. brasiliensis.

Several agents are known to induce differentiation in the human erythroleukemic cell line K562. In this work we have studied the ability of hyperthermia to induce differentiation in the K562 cell line. K562 cells were treated with hyperthermia in the range of 41-45 degrees C. Cell proliferation and the plating efficiency of heat treated cells along with their hemoglobin synthesis was measured and compared with controls. Hyperthermia severely inhibited the growth of K562 cells in the suspension culture in a time- and temperature-dependent manner. Sixty minutes of heating and 44 and 40 min of heating at 45 degrees C totally inhibited the growth of the cells. The number of clonogenic cells also decreased as a result of heat treatment. Extended periods of heating for more than 2 h at 41 degrees C resulted in thermal adaptation. Hyperthermia-induced hemoglobin synthesis by these cells, only at 42 and 43 degrees C. Maximum induction was observed after heat treatment for 80 min at 43 degrees C and 180 min at 42 degrees C. At lower temperature, although the fraction of surviving cells was high, but no signs of hemoglobin synthesis could be observed. At temperatures higher than 43 degrees C, the fraction of surviving cells decreased rapidly and also no signs of hemoglobin synthesis could be detected. At the two selective temperatures, hemoglobin synthesis started 4 days after heat treatment. The results showed that hyperthermia caused cytotoxicity and growth arrest and induced differentiation as judged by hemoglobin synthesis and reduced clonogenicity in this cell line. This is the first time that a physical agent has been shown to induce differentiation in erythroleukemic cell lines.

The eel pathogen Vibrio vulnificus biotype 2 comprises at least three serovars, with serovar E being the only one involved in both epizootics of eel vibriosis and sporadic cases of human infections. The virulent strains of this serovar (VSE) have only been recovered from clinical (mainly eel tissue) sources. The main objective of this work was to design and validate a new protocol for VSE-specific isolation from environmental samples. The key element of the new protocol is the broth used for the first step (saline eel serum broth [SEB]), which contains eel serum as a nutritive and selective component. This approach takes advantage of the ability of VSE cells to grow in eel serum and thus to separate themselves from the pool of competitors. The growth yield in SEB after 8 h of incubation was 1,000 times higher for VSE strains than for their putative competitors (including biotype 1 strains of the species). The selective and differential agar Vibrio vulnificus medium (VVM) was selected from five selective media for the second step because it gave the highest plating efficiency not only for the VSE group but also for other V. vulnificus groups, including biotype 3. The entire protocol was validated by field studies, with alkaline peptone water plus VVM as a control. V. vulnificus was isolated by both protocols, but serovar E was only recovered by the new method described here. All selected serovar E isolates were identified as VSE since they were virulent for both eels and iron-overloaded mice and resisted the bactericidal action of eel and iron-overloaded human sera. In conclusion, this new protocol is a suitable method for the isolation of VSE strains from environmental samples and is recommended for epidemiological studies of the pathogenic serovar E.

The purpose of this study was to improve the transduction efficiency of adenoviral vectors (Ad) in human CD34+ hematopoietic progenitor cells. CD34+ cells from cord blood or mobilized peripheral blood were incubated with tumor necrosis factor-alpha (TNF-alpha). After removal of free TNF-alpha, the cells were infected with an Ad encoding green fluorescent protein (GFP). One day later, viable cells were counted and analyzed for GFP and CD34 by flow cytometry. To visualize vectoral trafficking, CD34+ cells were incubated with fluorophore-conjugated Ad. Plating efficiencies of hematopoietic progenitors before and after transduction were evaluated by methylcellulose assays. Pretreatment with TNF-alpha increased the transduction efficiency more than twofold (39.2% versus 15.5%) in a dose-dependent manner and strongly improved the survival of GFP-positive CD34+ cells. Time course experiments showed that TNF-alpha incubation times as short as 10 minutes were still effective. Neutralizing antibodies to TNF receptor II and RGD peptides diminished the TNF-alpha-dependent increase in transduction efficiency. No TNF-alpha-dependent increase in adenoviral receptors (coxsackie-adenovirus receptor, alphavbeta3-integrin) occurred. Analysis of viral binding demonstrated a significantly higher incidence of local concentrations of Ad along the cell surface (caps) in virus-positive cells of the TNF-alpha-treated group. Plating efficiency, especially the formation of granulocyte-macrophage colony forming units, was enhanced by TNF-alpha pretreatment. We conclude that brief incubation with TNF-alpha before addition of the Ad significantly increased the Ad transduction efficiency in CD34+ cells, and improved post-transduction survival of progenitors of the granulocyte-macrophage lineage. This finding correlates with increased Ad capping at the cell surface and suggests an alteration of Ad trafficking.

An efficient and reproducible protocol is described for the regeneration of Astragalus melilotoides protoplasts isolated from hypocotyl-derived embryogenic calli. Maximum protoplast yield (11.74 +/- 0.6x10(5)/g FW) and viability (87.07 +/- 2.8%) were achieved using a mixture of 2% (w/v) Cellulase Onozuka R10, 0.5% (w/v) Cellulase Onozuka RS, 0.5% (w/v) Macerozyme R10, 0.5% (w/v) Hemicellulase, and 1% (w/v) Pectinase, all dissolved in a cell protoplast wash (CPW) salt solution with 13% (w/v) sorbitol. First divisions occurred 3-7 days following culture initiation. The highest division frequency (9.86 +/- 0.68%) and plating efficiency (1.68 +/- 0.05%) were obtained in solid-liquid medium (KM8P) supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 0.5 mg/l 6-benzylaminopurine (BA), 0.2 mg/l kinetin, 0.2 M glucose, 0.3 M mannitol and 500 mg/l casein hydrolysate. Upon transfer to MS medium with 0.5 mg/l alpha-naphthaleneacetic acid and 1-2 mg/l BA, the protoplast-derived calli produced plantlets via somatic embryogenesis (56.3 +/- 4.1%) and organogenesis (21.6 +/- 0.6%). Somatic embryos or adventitious shoots developed into well-rooted plantlets on MS medium without any plant growth regulators or supplemented with 3.0 mg/l indole-3-butyric acid, respectively. About 81% of the regenerants survived in soil, and all were normal with respect to morphology and growth characters.

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas 26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res. 63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.

Established cancer cell lines are useful in the study of various cancers. We established a human gastric carcinoma cell line TMC-1 derived from the lymph node of a moderately differentiated adenocarcinoma of the stomach. TMC-1 cells grew in vitro as a mixture of attached and suspension cells, and exhibited spindle or ovoid morphology. They had a population doubling time of 15 h, a plating efficiency of 61%, formed colonies in semisolid agar, secreted the tumor marker CA 19-9, and were tumorigenic in athymic nude mice. The cells expressed E-cadherin and β-catenin. The karyotypic analysis demonstrated hyperdiploid features with a modal chromosome of 53. The cell had the deletion at chromosome 18q and gains at chromosome 2p13–25, 5p15, 5q21–35, 7, 8q24, 9q, 11, 12p, 14q24–32 and 20. Analysis by fluorescence in situ hybridization showed the deletion at 7qtel and duplication at 7q11.2 at the rearranged chromosome 7. Growth of TMC-1 cells was inhibited by 27–32% by interferon-α (2,000 U/ml) and by interferon-γ with an IC50 of 125 U/ml. The cell line is tumorigenic in vivo, and its growth is moderately inhibited by interferon-α and interferon-γ. It can be used to develop new modalities of human gastric cancer treatment.

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw-1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non-ionic detergent as compared with wild-type cells. Tetrad analysis of crosses involving the cw-1 mutant confirmed 2:2 segregation of the cw:cw+ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw-1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw-1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.

The bystander effect on unirradiated neoplastic human salivary gland (HSGc) cells was investigated by co-culturing them with HSGc cells that had been irradiated with 290 MeV/u carbon beams of different linear energy transfer (LET) values. It was found that the plating efficiency and proliferation of the unirradiated recipient cells were increased and that these increases were related to the LET as well as the radiation dose. Exposure of HSGc cells to higher LET and higher dose was much more effective in enhancing the plating efficiency and proliferation of the unirradiated cells than exposure to lower LET and lower dose. However, when PTIO, a nitric oxide (NO)-specific scavenger, was present in the co-culture medium, the cell growth capacity of the unirradiated recipients was reduced to control level, indicating that NO is involved in the bystander response. As an oxidization product of NO, nitrite was detected in the co-culture medium and its concentration depended on the LET and dose of irradiation. Using a NO-generator sper/NO, it was verified that NO at low concentrations indeed enhanced cell proliferation. Accordingly, NO plays an important role in medium-mediated bystander effects.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by genomic instability, chronic oxidative damage, and increased cancer incidence. Compared to normal cells, AT cells exhibit unusual sensitivity to exogenous oxidants, including t-butyl hydroperoxide (t-BOOH). Since ferritin releases labile iron under oxidative stress (which is chronic in AT) and labile iron mediates the toxic effects of t-butyl hydroperoxide, we hypothesized that chelation of intracellular labile iron would increase the genomic stability of AT cells, with and without exogenous oxidative stress. Here we report that desferrioxamine treatment increases the plating efficiency of AT, but not normal cells, in the colony forming-efficiency assay (a method often used to measure genomic stability). Additionally, desferrioxamine increases AT, but not normal cell resistance, to t-butyl hydroperoxide in this assay. Last, AT cells exhibit increased sensitivity to the toxic effects of FeCl2 in the colony forming-efficiency assay and fail to demonstrate a FeCl2-induced G2 checkpoint response when compared to normal cells. Our data indicates that: (1) chelation of labile iron increases genomic stability in AT cells, but not normal cells; and (2) AT cells exhibit deficits in their responses to iron toxicity. While preliminary, our findings suggest that AT might be, in part, a disorder of iron metabolism and treatment of individuals with AT with desferrioxamine might have clinical efficacy.

Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at −80°C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.

Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 μg/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbecco's modified Eagle's medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 ± 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 μg/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 μg/ml) would be a useful cold preservation means for the development of cell therapies.

To identify genes that are frequently downregulated in hepatocellular carcinoma (HCC), a panel of putative underexpressed genes was first established by an in-house cDNA macroarray method. Two different assays, semiquantitative RT-PCR combined with Northern analysis and customized cDNA microarray analysis, were used to screen through these genes and the results were compared. Several genes, some with unknown function, were confirmed to be downregulated by both the methods. The effect of a downregulated gene, BMAL2, on cell proliferation was examined. Overexpression of antisense BMAL2 RNA in 293EBNA cells resulted in reduced cell cycle time, increased plating efficiency in soft agar, diminished TNF-alpha-induced increment of CPP32/caspase-3 activity, and a reduced proportion of cells in the G2 phase with a concomitantly increased proportion of cells in the S phase. In conclusion, by combining three different methods, we have obtained a panel of frequently down regulated genes in HCC, including BMAL2. Antisense overexpression of BMAL2 enhances cell proliferation.

Aims: To determine the reason that spores of Bacillus species, in particular Bacillus subtilis, are able to form colonies with high efficiency on media with very high salt concentrations. Methods and Results: Spores of various Bacillus species have a significantly higher plating efficiency on media with high salt concentration (termed osmoresistance) than do log or stationary phase cells. This spore osmoresistance is higher on richer media. Bacillus subtilis spores lacking various small, acid-soluble spore proteins (SASP) were generally significantly less osmoresistant than were wild-type spores, as shown previously (Ruzal et al. 1994). Other results included: (a) spore osmoresistance varied significantly between species; (b) the osmoresistance of spores lacking SASP was not restored well by amino acid osmolytes added to plating media, but was completely restored by glucose; (c) the osmoresistance of spores lacking SASP was restored upon brief germination in the absence of salt in a process that did not require protein synthesis; (d) significant amounts of amino acids generated by SASP degradation were retained within spores upon germination in a medium with high but not low salt; (e) slowing but not abolishing SASP degradation by loss of the SASP-specific germination protease (GPR) did not affect spore osmoresistance; (f) sporulation at higher temperatures produced less osmoresistant spores; and (g) spore osmoresistance was not decreased markedly by the absence of the stress sigma factor for RNA polymerase, σB. Conclusions: Spore osmoresistance appears as a result of three major factors: (1) specific characteristics of spores and cells of individual species; (2) the precise sporulation conditions that produce the spores; and (3) sufficient energy generation by the germinating and outgrowing spore to allow the spore to adapt to conditions of high osmotic strength; the substrates for this energy generation can come from either the endogenous generation of amino acids by SASP degradation or from the spore's environment, in the form of a readily taken up and metabolized energy source such as glucose. Signficance and Impact of Study: These results provide information on the mechanisms of spore osmoresistance, a spore property that can be of major applied significance given the use of high osmotic strength with or without high salt as a means of food preservation.

Schwann cell (SC) transplantation is a promising strategy for axonal regeneration in the nervous system. Identifying the grafted SCs is an important aspect of this approach. The current study sought to establish a simple, reliable, fluorescent labeling method for SCs with a lipophilic molecule, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). Human SCs were incubated with varying concentrations of CFSE for different time periods. Based on the viability of labeled SCs and its plating efficiency, 1 min incubation with 5 microM CFSE at 37 degrees C was selected as the optimal labeling condition. Flow cytometric analysis and fluorescence microscopy demonstrated that the fluorescence of labeled SCs would fade over 4 weeks. Immunostaining for the phenotypic expression of SC markers, including S100, GFAP, P75, and MHC-I/II at 1 and 4 weeks after incubation with CFSE showed no difference between labeled and non-labeled SCs. Mixed cultures of labeled human SCs and rat SCs for 48 h were performed in triplicate and demonstrated that no leakage of dye from labeled SCs in cell culture occurred across species. A total of 14 injections of 2x10(5) labeled SCs were performed within the spinal cord at T8 and/or L1 level in 9 nude rats. The animals were euthanized at 1 (6 injections) and 4 weeks (8 injections). Grafted labeled SCs survived for at least 4 weeks, and could be easily recognized in the nude rat spinal cord without leakage of dye to surrounding cells. The SCs migrated in white and gray matter 3-6 mm away from the injection and in the central canal for up to 12 mm. These results suggest that CFSE can be used as a fluorescent tracer of human SCs for both in vitro and in vivo studies, for a period of at least 4 weeks.

OBJECTIVE: The aim of our study was to use (1H) nuclear magnetic resonance (NMR) spectroscopy as a tool to assess metabolic functions of hepatocytes and to monitor major metabolic pathways of these cells during culture following hypothermic preservation. METHODS: After isolation, 2 x 10(7) primary porcine hepatocytes were preserved at 4 degrees C in supplemented Leibovitz L-15 medium for 48 h. Viability was assessed at isolation, 24 and 48 h. At isolation and at 48 h cells were plated and cultured with serum free supplemented Williams E medium. 1H NMR spectroscopy was used to assess indices of glucose metabolism, ammonia clearance indices and ketone bodies precursors at 48 h post-plating. Peak integration was applied with sodium 3-(trimethylsilyl-2,2,3,3-2H4)-1-propionate as an internal standard to obtain quantitative results. RESULTS: Results were obtained from six isolations. Viability was 78.1 +/- 1.2% at isolation, 69 +/- 3.4% at 24 h and 58.9 +/- 3.8% at 48 h of hypothermia. Plating efficiency was 87 +/- 4% for freshly isolated cells and 33.6 +/- 7.6% for hypothermically preserved cells. Glucose consumption was comparable in both groups. Hypothermically preserved cells consumed more threonine, produced more pyruvate and alanine but less lactate. They also produced less acetate and consumed less tyrosine. Glutamate and glutamine concentrations were similar under both conditions. CONCLUSION: 1H NMR spectroscopy is a valid method for assessing metabolic pathways of cultured primary porcine hepatocytes. Although hypothermically preserved cells had a reduced plating efficiency, they were still metabolically active. Thus, hypothermia can be used as a temporary preservation technique for primary porcine hepatocytes.

Culture conditions are described for sustained cell division and plant regeneration from protoplasts of rose (Rosa hybrida L. `Sumpath'). Protoplasts were enzymatically isolated from 2-week-old embryogenic cell suspension cultures. Freshly isolated protoplasts were plated as a thin layer onto protoplast culture medium (half-strength 21 Murashige and Skoog's medium containing 60 g l−1 myo-inositol, 4.4 μM BA, and 1.4 μM 2,4-D) at a density of 5×104 protoplasts ml−1. The plating efficiency reached 3.9% after 2 weeks of culture. However, few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l−1 myo-inositol was replaced with the same osmolarity of 90 g l−1 mannitol, indicating that myo-inositol is essential for sustained cell division of protoplasts. Colonies were formed after 8 weeks of culture at a frequency of 0.2%. Colonies were then transferred to colony culture medium (0.4% Gelrite-solidified protoplast culture medium) and maintained by subculturing at 4-week intervals to form embryogenic calluses. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Somatic embryos were transferred to half-strength MS basal medium containing 48 mg l−1 ferric ethylenediamine di-(o-hydroxyphenylacetate), where they subsequently developed into plantlets at a frequency of 30.9%. The plantlets had the same chromosome number of 2n=3x=21 as the source plant. They were successfully transplanted to potting soil and grown to maturity in a greenhouse.

A selective medium (designated as NSVC-In medium) was developed to isolate of Erwinia ananas, the causal agent of bacterial palea browning of rice. Medium NSVC-In contains per 1000ml of distilled water: 10g inositol, 10g peptone, 50g NaCl, 100mg vancomycin, 100mg cycloheximide, and 15g agar. The medium is adjusted to pH 6.8 with NaOH before autoclaving. Plant pathogenic E. ananas grew well on this medium to develop characteristic colonies, but the growth of nonpathogenic E. ananas and Erwinia herbicola was inhibited. Plating efficiency of this medium for pathogenic E. ananas was the same as that of nutrient agar medium. Pathogenic E. ananas could be selectively detected from dead leaves of rice plants.

protoplasts was developed for the Liliaceous ornamental plant, Agapanthus praecox ssp. orientalis (Leighton) Leighton `Royal Purple Select' (2n=2x=32).Viable protoplasts were routinely isolated from leaf-derived embryogenic calluses with yields of 0.8 to 1.5x10 protoplasts per g FW of calluses. Protoplasts started to divide 5 to 7 days after isolation, and protoplast-derived colonies consisting of 50 to 100 cells were obtained after 1 month. A plating efficiency of 0.8% was obtained after 2 months of culture using a gellan gum-solidified medium containing 1 mg 1-1 each of PIC and BA under continuous illumination. Protoplastderived calluses produced somatic embryos at a frequency of 46.7 % on PGR-free medium, whereas 68.3 % of the calluses regenerated adventitious shoots on a medium containing 1 mg 1-1 BA. Somatic embryos and adventitious shoots developed into plantlets, which were successfully transplanted to pots. Flow cytometric analysis and chromosome observation revealed that both diploid and tetraploid plants were regenerated from protoplasts.