Prolonged Viability Research Articles

Studies on the pollen biology of Terminalia paniculata Roth. (Combretaceae)

Terminalia paniculata is a deciduous tree widely distributed in tropical semi-evergreen and moist deciduous forest. Flowers of T. paniculata have ten stamens, which remain inside the bud and anthesis is carried out at different times of the day. Pollen grains are medium spherical, tricolpate 17 mm in diameter, smooth exine and yellow in colour. The pollen: ovule ratio approximated 16,000: 1. Pollination efficiency was increased daily, from first day of flower opening (0.000076), reaching the maximum on third day (0.00083) and there was no pollen deposition on the fourth day. To effectively assess in-vitro pollen viability in T. paniculata, an optimized germination medium (30% sucrose with Brewbaker and Kwak’s medium) was developed. Pollen grains stored under cryopreservation (-20oC) had prolonged viability. Using the hand pollination for testing the in-vivoviability of pollen grains the maximum number of fruit set was obtained in the fresh pollen grains (0 h – pollen collected at the time of anthesis) on the stigma. Afterwards, increase in the age of the pollen decreased the fruit set. After 15 h of anthesis, the pollen grains lost their viability and there was no fruit set. The current findings will be useful in studying, pollen – pistil interactions, gene flow and heterozygosity of the T. paniculata populations. Key words: Cryopreservation, in vitro germination, pollen storage.

Beneficial effect of 30Kc6 gene expression on production of recombinant interferon-β in serum-free suspension culture of CHO cells

The Bombyx mori 30Kc gene is known to have anti-apoptotic activity and can enhance the cell growth and expression of recombinant proteins in anchorage-dependent CHO cell cultures. In this study, an interferon-β (IFN-β)-producing CHO cell line, which expresses the recombinant 30Kc6 gene, was constructed to investigate the effect of 30Kc6 expression on the production of IFN-β in serum-free suspension culture. The 30Kc6 expressing cell line showed lower apoptotic activity and prolonged cell viability under apoptotic conditions induced by the addition of sodium butyrate, staurosporine, or the removal of serum. The 30Kc6 expressing cell line also suppressed the loss of mitochondrial membrane potential induced under these conditions. It was observed that viability, and production of IFN-β were also enhanced by 30Kc6 expression in serum-free suspension cultures. These results indicate that the 30Kc6 gene can positively affect the viability and production of recombinant therapeutic proteins in serum-free suspension cultures of CHO cell lines.

Metabolic Rates, Growth Phase, and mRNA Levels Influence Cell-Specific Antibody Production Levels from In Vitro-Cultured Mammalian Cells at Sub-Physiological Temperatures

Previous work has shown that recombinant protein yield can be improved from in vitro-cultured mammalian cells by culturing at sub-physiological temperatures, although this effect is cell line and product dependent. The mechanism(s) by which low temperature leads to enhanced product yield are currently unknown; however, recent reports suggest that increased mRNA levels at sub-physiological temperatures may be largely responsible for this. Here, we have investigated whether low-temperature cultivation of cell lines selected for antibody production at 37 degrees C leads to changes in heavy- and light-chain mRNA levels and if this is reflected in antibody yields. Low-temperature in vitro culturing resulted in reduced viable cell concentration, prolonged cell viability, a reduction in metabolite consumption and production, cell cycle arrest in both CHO and NS0 cells, and changes in the levels of heavy- and light-chain mRNA. Despite increases in the level of heavy- and light-chain mRNA upon culturing at 32 degrees C in our model CHO cell line, this did not result in increased total product yield; however, changes in cell-specific yields were observed that reflected the metabolic rate of glucose utilization and changes in mRNA levels.

Alginate-based microencapsulation of retinal pigment epithelial cell line for cell therapy

The goals of this study were to evaluate human retinal pigment epithelial cell line (ARPE-19) for cell encapsulation and to optimize the alginate-based microencapsulation. We used immortalized ARPE-19 cells and the transfected sub-line that expresses secreted alkaline phosphatase (SEAP) reporter enzyme. Alginate was cross-linked with different divalent cations (Ca 2+, Ba 2+, Sr 2+ and combination of Ca 2+ and Ba 2+), coated first with poly- l-lysine (PLL), and then with alginate. Microcapsules with different pore sizes and stability were generated. The pore size of the microcapsules was assessed by the release of encapsulated fluorescein isothiocyanate (FITC)-dextrans. The viability of the cells in the microcapsules was studied in vitro by assessing the secretion rates of SEAP and oxygen consumption by the cells. The best microcapsule morphology, durability and cellular viability were obtained with alginate microcapsules that were cross-linked with Ca 2+ and Ba 2+ ions and then coated with PLL and alginate. Based on FITC-dextran release these microcapsules have porous wall that enables the rapid contents release. The ARPE-19 cells maintained viability in the Ca 2+ and Ba 2+ cross-linked microcapsules for at least 110 days. The alginate microcapsules cross-linked with Ca 2+ and Ba 2+ have sufficiently large pore size for prolonged cell viability and for the release of secreted SEAP model protein (Mw 50 kDa; radius of gyration of 3 nm). ARPE-19 cells show long-term viability and protein secretion within alginate microcapsules cross-linked with Ca 2+ and Ba 2+. This combination may be useful in cell therapy.

Short Peptides Enhance Single Cell Adhesion and Viability on Microarrays

Single cell patterning holds important implications for biology, biochemistry, biotechnology, medicine, and bioinformatics. The challenge for single cell patterning is to produce small islands hosting only single cells and retaining their viability for a prolonged period of time. This study demonstrated a surface engineering approach that uses a covalently bound short peptide as a mediator to pattern cells with improved single cell adhesion and prolonged cellular viability on gold patterned SiO2 substrates. The underlying hypothesis is that cell adhesion is regulated by the type, availability, and stability of effective cell adhesion peptides, and thus covalently bound short peptides would promote cell spreading and, thus, single cell adhesion and viability. The effectiveness of this approach and the underlying mechanism for the increased probability of single cell adhesion and prolonged cell viability by short peptides were studied by comparing cellular behavior of human umbilical cord vein endothelial cells on three model surfaces whose gold electrodes were immobilized with fibronectin, physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently bound Lys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and binding properties were characterized by reflectance Fourier transform infrared spectroscopy. Both short peptides were superior to fibronectin in producing adhesion of only single cells, whereas the covalently bound peptide also reduced apoptosis and necrosis of adhered cells. Controlling cell spreading by peptide binding domains to regulate apoptosis and viability represents a fundamental mechanism in cell-materials interaction and provides an effective strategy in engineering arrays of single cells.

In PC12 Cells Neurotoxicity Induced by Methamphetamine Is Related to Proteasome Inhibition

Neurodegenerative disorders are featured by a variety of pathological hallmarks, and very often they are characterized by neuronal inclusions in specific brain nuclei. Occurrence of neuronal inclusions has been often related to the onset of cell death. Recent studies demonstrated that amphetamine derivatives produce intracellular inclusions, which are reminiscent of those occurring in degenerative disorders. In the present article, we analyzed the correlation between neuronal inclusions and cell death using methamphetamine (METH) in PC12 cell cultures. We found that the dose necessary to induce cell death is higher compared with that required to induce inclusions formation. Our results demonstrate a dissociation between formation of inclusion bodies and cell death suggesting that neuronal inclusions do not necessarily lead to cell death. The conclusions of the present article suggest that the onset of inclusion bodies represents a slight consequence of toxicity, which requires a prolonged cell viability to take place.

Cell-Directed Assembly of Lipid-Silica Nanostructures Providing Extended Cell Viability

Amphiphilic phospholipids were used to direct the formation of biocompatible, uniform silica nanostructures in the presence of Saccharomyces cerevisiae and bacterial cell lines. The cell surfaces organize multilayered phospholipid vesicles that interface coherently with the silica host and help relieve drying stresses that develop with conventional templates. These host structures maintain cell accessibility, addressability, and viability in the absence of buffer or an external fluidic architecture. The cell surfaces are accessible and can be used to localize added proteins, plasmids, and nanocrystals. Prolonged cell viability combined with reporter protein expression enabled stand-alone cell-based sensing.

C5a Receptor Deficiency Attenuates T Cell Function and Renal Disease in MRLlpr Mice

The development and progression of systemic lupus erythematosus (SLE) is strongly associated with complement activation and deposition. To characterize the role of C5a and its receptor (C5aR) in SLE, C5aR-deficient mice were backcrossed nine generations onto the lupus-like MRL(lpr) genetic background. Evidence is presented that C5aR modulates both renal injury and T cell responses in MRL(lpr) mouse. C5aR-deficient MRL(lpr) mice had prolonged viability, with a mean survival time of 33.0 wk compared with 22.6 wk in control mice. Renal injury was also attenuated in the C5aR-/- MRL(lpr) mice. At 20 wk of age C5aR-/- MRL(lpr) mice had a complete absence of glomerular crescents and marked reductions in glomerular hypercellularity. There was no difference in the degree of glomerular C3 deposition; however, IgG deposits were reduced in the C5aR-/- MRL(lpr) mice. The reduction in glomerular injury was also associated with a four-fold decrease in renal CD4+ T cell infiltrates. Whereas there were modest differences in total IgG anti-dsDNA antibody titers, C5aR-deficient mice had 3.5-fold higher levels of IgG1 and 15-fold lower levels of IgG2a anti-dsDNA antibody titers compared to controls. The differences in anti-dsDNA IgG subclasses were associated with reduced CD4+ Th-1 responses in the C5aR-/- MRL(lpr) mice, including diminished production of IL-12p70, IFN-gamma, and increased expression of the Th-2 transcription factor GATA-3. These findings indicate that the C5aR plays a major role in modulating complement-dependent renal injury and T helper cell Th-1 responses in the MRL(lpr) mouse.

61. Higher In Vivo Maintenance, Biodistribution and Immunopotency of Dendritic Cell Vaccines Self-Differentiated through Lentiviral Vector Programming

Top of pageAbstract Dendritic cell (DC) vaccines are conventionally produced ex vivo by culturing DC precursors in the presence of recombinant growth factors. Methods to facilitate the generation of DCs, to prolong their viability, to facilitate their migration to lymphoid tissues and to augment their capacity to establish and maintain antigen-specific immune responses are critical for improving their effective therapeutic potential for immunotherapy. To address these issues, we have genetically engineered purified human CD14+ monocytes with lentiviral vectors (LV) for production of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), which triggered their differentiation into cells with typical characteristics of DCs. We previously demonstrated that these lentivirus programmed DCs (DC/LVs) exhibited more potent immunostimulatory function and prolonged viability in vitro than DCs generated by conventional methods. Using the same strategy, we have now generated murine DC/LVs in order to test the efficacy of this approach in vivo. Third generation self-inactivating LV (5 |[mu]|g/ml p24 equivalent) transduced non-adherent bone marrow (BM) cells harvested from C57BL/6 mice at >90% transduction efficiency. BM cells co-expressing GM-CSF and IL-4 after LV transduction produced these cytokines at concentrations similar to those commonly used for generation of conventionally cultured DCs by addition of exogenous cytokines (> 10ng/ml), resulting in the generation of DC/LVs as evidenced by their morphology and immunophenotype (CD11b+, CD11c high, CD80 high, MHCII high). DC/LVs maintained in culture were metabolically active and viable for 3 weeks, whereas conventional DCs showed a drastic decrease in viability in the first week of culture. In vivo bioluminescence imaging studies were then performed using luciferase as a marking gene to track DCs after s.c. injection into nude syngeneic mice. Persistent and robust bioluminescence signals were detectable from the injected DC/LVs for more than 45 days, whereas signals produced by conv. DCs faded away within the first two weeks. To determine whether the higher viability of DC/LVs correlated with greater potency as vaccines, we next examined their antigen presentation and immunostimulatory functions in vivo. DC/LVs and conv. DCs maintained in culture for 7 days were transduced to express the melanoma associated antigen MART-1 and injected s.c. Splenocytes harvested 2 weeks after vaccination with DC/LVs showed up to 2-fold increase in activated T cells by IFN|[gamma]|- ELISPOT assay (p<0.05), and chromium lysis assays using target B16 melanoma cells showed more efficient killing by DC/LV-activated CTLs in comparison to conv. DCs (p<0.05). We are currently evaluating the immunotherapeutic efficacy of DC/LVs injected into mice developing B16 melanoma tumors. Initial experiments in a prophylactic model indicate that mice vaccinated with DC/LV show enhanced survival compared to the group vaccinated with conv.DCs. Our results therefore indicate that DC/LVs can provide enhanced anti-tumoral therapeutic efficacy and warrant further investigation of the clinical applicability of genetically programmed DCs.

Signal transduction and cellular functions of the TEL/ARG oncoprotein

The TEL/ARG oncogene is formed by t(1;12)(q25;p13) reciprocal translocation and is associated with human leukemia. We have previously demonstrated that the expression of TEL/ARG in Ba/F3 cells results in prolonged viability and hyper-responsiveness to IL-3. To determine the molecular mechanisms, a series of mutants of TEL/ARG were generated, and each cDNA was expressed in Ba/F3 or CHO cells. The PNT domain in TEL and K317 in ARG were essential for both signaling and biological effects. The SH3 domain in ARG was required for hyper-responsiveness to IL-3, but not for prolonged viability. The opposite was true for the SH2 domain in ARG. Mutation of Y314 in TEL, a putative GRB2-binding site, led to reduced viability, and loss of hyper-responsiveness to IL-3. All biological functions were profoundly impaired with deletion of the C-terminus in ARG, despite maintaining high levels of its kinase activity. When expressed in CHO cells, wild-type TEL/ARG induced the formation of fillopodia, in a fashion dependent on the C-terminal portion and intact kinase activity. Thus, these results suggest several critical domains within TEL/ARG necessary for function, and indicate that the signaling pathways necessary for viability, growth factor hyper-responsiveness and cytoskeletal reorganization are likely to be separate.

Effects of storage and incubation temperature on the viability of eggs, embryos and larvae in two strains of an African catfish, Heterobranchus longifilis (Siluriformes, Clariidae)

Two experiments, dealing with short-term storage of ova and thermal conditions to optimize gamete and eggs management in hatcheries of the African catfish, Heterobranchus longifilis, were carried out. In the first experiment, ova collected by stripping from two strains of H. longifilis were stored for intervals up to 8 h at two temperature regimes: in a domestic refrigerator (3–5°C) and at ambient room temperature (20.5–22°C). In the second experiment, eggs were incubated from fertilization to hatching at different experimental temperatures (21, 25, 29, 32 and 35°C) to determine the effects of temperature on the kinetics of white egg appearance, hatching times and hatching quality. Gamete storage at warmer temperatures significantly prolonged viability irrespective of the strain used. In fact, the hatching rate for ova stored at 20.5–22 and 3–5°C for 5 h ranged between 75.2–79.3% and 6.5–9.4% respectively. Loss of viability was most noticeable after 6 h storage at ambient room temperature. Post-storage viability significantly declined after 2 h exposure to the domestic refrigerator temperature. No hatching of normal larvae took place after 8 h post-storage time. Results from the second experiment showed that time to maximum whitening of eggs was both strain- and temperature-dependent. The time to maximum mortality of eggs was shorter in the Layo strain (LS) than in the Noun strain (NS), regardless of incubation temperature. The appearance of white eggs was shorter with increasing incubation temperatures. Hatching times decreased with increasing temperature, regardless of strain. Hatching took place from 21 to 27 h and 19 to 24 h after fertilization at temperature of 29°C, respectively, for NS and LS. The length of the hatching period was remarkably shorter for LS than NS at any tested incubation temperature, except 35°C. No hatching took place at 21°C. The highest proportion of normal larvae occurred at 25 and 29°C, respectively, for NS and LS. Hatching rate was highest at 25 and 29°C, respectively, for NS and LS. There was a significantly higher proportion of deformed larvae at 35°C regardless of the strain.

The experimental antitumor agents Phortress and doxorubicin are equiactive against human-derived breast carcinoma xenograft models.

Phortress (the dihydrochloride salt of the lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluoro-benzothiazole (5F 203)) is an experimental antitumor agent with potent and selective activity against human-derived carcinomas of breast, ovarian and renal origin. UK clinical trials of Phortress are scheduled to begin in 2004. The mechanism of action of Phortress is distinct from all classes of chemotherapeutic agents currently in the clinic, and involves metabolic activation by cytochrome P450 (CYP) 1A1 to electrophilic species, which generate DNA adducts in sensitive tumors only. In the present study, the antitumor efficacy of Phortress has been compared with that of doxorubicin (Dox) in nine human-derived mammary carcinoma xenograft models, cultivated subcutaneously in the flanks of nude mice. In addition, cyp1a1 mRNA expression was measured in tumors of control and treated animals. Phortress compared favorably with Dox: significant activity, independent of estrogen receptor (ER) status, was established in 7/9 xenografts; in one xenograft model, Phortress elicited superior antitumor activity; no model demonstrated complete resistance to Phortress. In accordance with this observation, all xenografts available for examination (8) displayed clear induction of cyp1a1 expression upon treatment of mice with Phortress whereas Dox failed to induce cyp1a1 expression in all models. Prolonged viability of tumor fragments, recovered for treatment ex vivo could not be sustained; thus correlations between tumor cells' response to Phortress and cyp1a1 or cyp1b1 inducibility following 5F 203 treatment could not be determined with confidence.

Genetic analysis of Treponema denticola ATCC 35405 biofilm formation.

Treponema denticola is a major aetiological organism implicated in periodontal disease. The interaction of T. denticola with other oral bacteria, in particular Porphyromonas gingivalis, in biofilm formation is thought to be an important step in the onset of periodontal disease. The interaction between T. denticola and P. gingivalis has been examined using a panel of T. denticola mutants and their effects on mixed biofilm formation tested in a static biofilm model. T. denticola ATCC 35405 did not form detectable biofilms on various inert surfaces. However, the spirochaete was demonstrated to form a biofilm with preattached P. gingivalis 381. T. denticola cfpA, which lacks the cytoplasmic filament, was unable to produce a mixed biofilm with P. gingivalis. A T. denticola flgE mutant which lacks the flagella hook protein and is therefore non-motile displayed a reduced, but readily detectable, ability to form a mixed biofilm as did the T. denticola mutant which does not possess the major outer sheath protein (Msp). The T. denticola lrrA mutant was only moderately defective in forming mixed biofilms with P. gingivalis. However, the T. denticola methyl-accepting chemotaxis protein (DmcA) did not appear to play a major role in mixed biofilm formation. In contrast, T. denticola lacking the PrtP protein for prolyl-phenylalanine-specific protease, showed an increased ability to form mixed biofilms and a prolonged viability in the biofilm.

Thermobiochemical evidence for the rapid metabolic rate in hybridoma cells genetically engineered to overexpress the anti-apoptotic protein bcl-2 in batch culture

A serious problem in the culture of animal cells to produce therapeutic proteins is apoptosis (programmed cell death) because it restricts the ability of the cell culture to yield the heterologous material. One means to delay apoptosis is genetically to engineer the anti-apoptotic gene bcl-2 into the genome. This had been adopted elsewhere to give TB/C3 hybridoma cells that overexpressed bcl-2 (pEF bcl-2-MC1neopA plasmid) and a control, pEF, which contained a nonsense sequence (pEF-MC1neopA). In the spinner cultures used in the present investigation, bcl-2 cells grew for 60 h to give greater cell density at a faster specific rate and with prolonged better viability than the controls in which, after 36 h, the membrane integrity and the apoptotic index of the cells declined rapidly leading to death. Prior to 36 h, production of the monoclonal antibody was only slightly higher in the pEF control giving little evidence for the metabolic burden of its production on antibody synthesis. Bcl-2 synthesis, however, was associated with 125% increase in heat flow rate (HFR) measured in the chemically (triacetin) calibrated batch microcalorimeter. HFR is a function of the metabolic rate and it is suggested that its greater level in the bcl-2 cells than the control was caused by the increased protein production due to the bcl-2 gene expression. The bcl-2 cells had an increased lactate flux compared with the control. The calorimetric–respirometric (CR) ratio confirmed the likelihood that glycolysis rather than glutaminolysis was the primary reason for this increase. It may be due to the limitation in mitochondrial capacity and/or the paucity of biosynthetic precursors leading to production of them from glucose. The stationary liquid phase balance (SLPB) was shown to be the appropriate on-line method to measure the oxygen uptake rate (OUR) in the dilute cell suspensions grown in the tank bioreactor. The metabolic rate expressed as HFR was measured in a customised flow microcalorimeter, which had been calibrated using the enthalpy of hydrolysis produced by the new test reaction using methyl paraben. Both HFR and OUR declined before the decrease in cell density in batch culture, indicating that they were sensitive indicators of deteriorating environmental conditions. However, neither proved to be an early detector of the onset of apoptosis.

Functional and morphological characteristics of bovine adrenal chromaffin cells on macroporous poly(D,L-lactide-co-glycolide) scaffolds.

Adrenal chromaffin cells (ACCs) secrete several neuroactive substances that are effective in influencing pain sensitivity in the central nervous system as well as enhancing the recovery of the intrinsic nigrostriatal dopaminergic system in patients with Parkinson's disease. ACC transplantation may be upregulated by the use of three-dimensional (3-D) scaffolds. In this study, we determined whether biodegradable poly(D,L-lactic-coglycolic acid) (PLGA) (85:15) sponges could be used as support for chromaffin cells. ACCs were isolated from bovine adrenal glands by standard perfusion (95% purity) followed by additional purification (>99.5% purity). ACC (approximately 5 x 10(5) cells) suspension in collagen (type I) was seeded on prewetted sponges and cultured in DMEM-F12 (1:1) medium (5% fetal bovine serum). The catecholamine and enkephalin levels of the samples were measured by high-performance liquid chromatography and radioimmunoassay. Cell morphology was examined by transmission electron microscopy. Morphological evidence showed prolonged viability of chromaffin cells on scaffolds having pores of 250-400 microm. Cell counts and scanning electron microscopy demonstrated that the majority of seeded cells were located within the scaffold. Chromaffin cells exhibited higher levels of enkephalins and catecholamines on PLGA scaffold compared with their monolayer cultures. By the use of 3-D PLGA as support for ACCs, it is possible to upregulate metabolic function and localize a high number of morphologically healthy-looking cells. Highly purified ACCs cultured on PLGA scaffold may have promise in transplantation studies, because these cells are less immunogenic and may be applied to in vivo settings by using short-term immunosuppression.

Survival of Filamentous Fungi in Hypersaline Dead Sea Water

A variety of filamentous fungi have recently been isolated from the Dead Sea (340 g/L total dissolved salts). To assess the extent to which such fungi can survive for prolonged periods in Dead Sea water, we examined the survival of both spores and mycelia in undiluted Dead Sea water and in Dead Sea water diluted to different degrees with distilled water. Mycelia of Aspergillus versicolor and Chaetomium globosum strains isolated from the Dead Sea remained viable for up to 8 weeks in undiluted Dead Sea water. Four Dead Sea isolates (A. versicolor, Eurotium herbariorum, Gymnascella marismortui, and C. globosum) retained their viability in Dead Sea water diluted to 80% during the 12 weeks of the experiment. Mycelia of all species survived for the full term of the experiment in Dead Sea water diluted to 50% and 10% of its original salinity. Comparison of the survival of Dead Sea species and closely related isolates obtained from other locations showed prolonged viability of the strains obtained from the Dead Sea. Spores of isolates obtained from the terrestrial shore of the Dead Sea generally proved less tolerant to suspension in undiluted Dead Sea water than spores of species isolated from the water column. Spores of the species isolated from the control sites had lost their viability in undiluted Dead Sea water within 12 weeks. However, with the exception of Emericella spores, which showed poor survival, a substantial fraction of the spores of Dead Sea fungal isolates remained viable for that period. The difference in survival rate between spores and mycelia of isolates of the same species points to the existence of adapted halotolerant and/or halophilic fungi in the Dead Sea.

EBNA3A association with RBP-Jkappa down-regulates c-myc and Epstein-Barr virus-transformed lymphoblast growth.

Epstein-Barr virus nuclear antigen protein 3A (EBNA3A) is one of four EBNAs (EBNA-2, EBNALP, EBNA3A, and EBNA3C) through the cellular DNA sequence-specific transcription factor RBP-Jkappa/CBF-1/CSL and are essential for conversion of primary B lymphocytes to lymphoblastoid cell lines (LCLs). In the present study, we investigated the effects of EBNA3A on EBNA2 activation of transcription in the IB4 LCL by conditionally overexpressing EBNA3A three- to fivefold. EBNA3A overexpression increased EBNA3A association with RBP-Jkappa, did not change EBNA3C association with RBP-Jkappa or EBNA or LMP1 expression, decreased EBNA2 association with RBP-Jkappa, decreased c-myc expression, and caused G(0)/G(1) growth arrest with prolonged viability. Expression of the fusion protein MycERTM in cells with conditional EBNA3A overexpression restored cell cycle progression and caused apoptosis. In contrast, MycER in the same cells without EBNA3A overexpression enhanced cell proliferation and did not increase apoptosis. These data indicate that EBNA3A overexpression inhibits protection from c-myc-induced apoptosis. In assays of EBNA2- and RBP-Jkappa-dependent transcription, EBNA3A amino acids 1 to 386 were sufficient for repression equivalent to that by wild-type EBNA3A, amino acids 1 to 124 were unimportant, amino acids 1 to 277 were insufficient, and a triple alanine substitution within the EBNA3A core RBP-Jkappa binding domain was a null mutation. In reverse genetic experiments with IB4 LCLs, the effects of conditional EBNA3A overexpression on c-myc expression and proliferation did not require amino acids 524 to 944 but did require amino acids 278 to 524 as well as wild-type sequence in the core RBP-Jkappa binding domain. The dependence of EBNA3A effects on the core RBP-Jkappa interaction domain and on the more C-terminal amino acids (amino acids 278 to 524) required for efficient RBP-Jkappa association strongly implicates RBP-Jkappa in c-myc promoter regulation.

Bcl-2-mediated regulation of CD69-induced apoptosis of human eosinophils: identification and characterization of a novel receptor-induced mechanism and relationship to CD95-transduced signalling.

Elimination of the eosinophils from the airways by selective induction of apoptosis represents a therapeutic approach for asthma. Here we report on a possible target molecule, the surface receptor CD69. To simulate an asthmatic response, segmental allergen challenge in mild asthmatics was performed. Eosinophil numbers increased in bronchoalveolar lavage (BAL) at 18 h. In contrast to blood cells, BAL eosinophils expressed the activation marker CD69. Purified blood eosinophils stimulated with granulocyte/macrophage colony-stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma) expressed CD69 and showed prolonged viability. Only IFN-gamma enhanced constitutive CD95 expression. Coincubation with anti-CD69 or anti-CD95 monoclonal antibody (MoAb) induced apoptosis, as revealed by propidium iodide incorporation, membrane blebbing and nuclear fragmentation. Additionally, both anti-CD69 and anti-CD95 MoAb reduced cytokine-enhanced Bcl-2 expression. In conclusion, CD69 transduces a Bcl-2-dependent death signal when ligated by a specific antibody. As, in contrast to the ubiquitous death-inducer CD95, the function of CD69 appears to be restricted to activated eosinophils, it represents an ideal target for therapeutic intervention in asthma.

Changes in macrophage morphology and prolonged cell viability following exposure to polyethylene particulate in vitro.

The interaction of macrophages and ultra-high molecular weight polyethylene (PE) wear plays an important role in perpetuating chronic inflammation at the bone implant interface, leading to peri-implant osteolysis and mechanical failure of the implant. A model to study the interaction of human mature macrophages with orthopaedic biomaterial wear has been previously developed. With the use of the model, in this study, the mature human monocyte-derived macrophages (MDMs) were observed with light, fluorescent, and scanning electron microscopy (SEM), as well as transmission electron microscopy (TEM). The cell viability was investigated using calcein and ethidium staining. Following exposure to PE particulate, the morphology of the human MDMs was heterogeneous: rounded, flattened, and elongated. There was no morphological evidence of cytotoxicity or apoptosis. The MDM viability was not influenced by phagocytosis of PE particulate in a negative fashion. In fact, more prolonged cell viability was observed in the human MDMs exposed to PE particulate when compared to controls.

Preservation of Nostocacean Hormogone Motility in Desiccated Calcium Carbonate Agar Patches

AbstractFilamentous nitrogen fixing cyanobacteria are resistant to desiccation, nutrient depletion, adverse temperature and challenging photic environments. This is generally thought to account for their role as pioneering forms of life on denuded strata. It has also lent credence to the possibility that cyanobacteria with prolonged viability may have been able to survive conditions of interplanetary space to implement a panspermian hypothesis for the origin of life on earth. Prolonged retention of viability had been noted in our collection of myxotrophically grown strains of nostocaceans cultured in liquid or solid media containing calcium carbonate. Such media permitted satisfactory recovery and regrowth after storage at low light intensities for 2 years or more. Moreover, even the oldest calcium carbonate cultures contained motile hormogonia when observed microscopically. This phenomenon can be used to advantage in sending cultures to other laboratories. Not only are organisms maintained during passage through the postal system but since the cultures formed hormogonia readily, it was possible to produce preservable homogeneous agar lawns that generated hormogonia as needed for demonstration or experiment. A practical technique consists of seeding axenic strains of Nostoc species grown in liquid shake culture under cool‐white fluorescent illumination into solid media containing 0.7% sucrose, 0.05% finely divided calcium carbonate and 1.4% purified agar. The preparations are incubated under red fluorescent illumination to produce several crops of hormogonia in successive cycles of development, within the agar. The heavily grown lawns are cut into disks or squares, then transferred to dishes and dried in desiccators with anhydrous calcium sulfate. The dry patches produce swarms of hormogonia in 2 or 3 h, depending upon the rate of rehydration. The patches are facile tools for purifying cultures through the isolation of hormogonia and studying cyanobacterial physiology by the rapid response of motile hormogonia.