Antiproliferative effects of two hydrosoluble derivatives of oxysterols on cultured lymphoma cells.

The antitumor effects of two new hydrosoluble derivatives of oxygenated sterols: JB69 and JC40 have been evaluated in vitro on a panel of lymphoma and leukemia cells from human and murine origins. These compounds result from the combination of a nucleotide with 7 beta-hydroxycholesterol (JB69) or 7 beta,25-dihydroxycholesterol (JC40). Both derivatives exhibit a significant cytotoxic activity against the different tumor cell lines tested, with some degree of difference between them. On the whole, the concentrations needed to inhibit the cell growth were found to be higher than those required for their parent compounds. However, two interesting features appeared in our experiments. (1) In a serum-free culture medium, cell lysis occurred within the first hours of incubation and seemed to result from the detergent-like properties possessed by this type of compounds. (2) In a culture medium supplemented with serum, we noted, that at high concentrations of JB69 (40 microM or 20 microM) and only with this oxysterol derivative, an important increase of incorporation of tritiated thymidine and uridine into DNA and RNA by viable cells. The origin of this effect is as yet unknown, but it strongly suggests a possible action on nucleic acids synthesis and metabolism. Taken together, these results emphasize the diversity and the complexity of the mechanisms involved in the cytotoxicity of these derivatives of oxysterols.     

A novel alginate hollow fiber bioreactor process for cellular therapy applications

Gel‐matrix culture environments provide tissue engineering scaffolds and cues that guide cell differentiation. For many cellular therapy applications such as for the production of islet‐like clusters to treat Type 1 diabetes, the need for large‐scale production can be anticipated. The throughput of the commonly used nozzle‐based devices for cell encapsulation is limited by the rate of droplet formation to ～0.5 L/h. This work describes a novel process for larger‐scale batch immobilization of mammalian cells in alginate‐filled hollow fiber bioreactors (AHFBRs). A methodology was developed whereby (1) alginate obstruction of the intra‐capillary space medium flow was negligible, (2) extra‐capillary alginate gelling was complete and (3) 83 ± 4% of the cells seeded and immobilized were recovered from the bioreactor. Chinese hamster ovary (CHO) cells were used as a model aggregate‐forming cell line that grew from mostly single cells to pancreatic islet‐sized spheroids in 8 days of AHFBR culture. CHO cell growth and metabolic rates in the AHFBR were comparable to small‐scale alginate slab controls. Then, the process was applied successfully to the culture of primary neonatal pancreatic porcine cells, without significant differences in cell viability compared with slab controls. As expected, alginate‐immobilized culture in the AHFBR increased the insulin content of these cells compared with suspension culture. The AHFBR process could be refined by adding matrix components or adapted to other reversible gels and cell types, providing a practical means for gel‐matrix assisted cultures for cellular therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Activity of recombinant cysteine-rich domain proteins derived from the membrane-bound MUC17/Muc3 family mucins

Background

The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and have cytoprotective properties. Their extracellular regions contain two EGF-like Cys-rich domains (CRD1 and CRD2) connected by an intervening linker segment with SEA module (L), and may function to stimulate intestinal cell restitution. The purpose of this study was to determine the effect of size, recombinant host source, and external tags on mucin CRD1-L-CRD2 protein activity.

Methods

Four recombinant Muc3-CRD proteins and three MUC17-CRD proteins were generated using Escherichiacoli or baculovirus-insect cell systems and tested in colonic cell cultures for activity related to cell migration and apoptosis.

Results

N-terminal glutathione-S-transferase (GST) or C-terminal His₈ tags had no effect on either the cell migration or anti-apoptosis activity of Muc3-CRD1-L-CRD2. His-tagged Muc3-CRD1-L-CRD2 proteins with truncated linker regions, or the linker region alone, did not demonstrate biologic activity. The human recombinant MUC17-CRD1-L-CRD2-His₈ was shown to have anti-apoptotic and pro-migratory activity, but did not stimulate cell proliferation. This protein showed similar in vitro biologic activity, whether produced in E. coli or a baculovirus-insect cell system.

Conclusions

Recombinant mucin proteins containing a bivalent display of Cys-rich domains accelerate colon cell migration and inhibit apoptosis, require a full-length intervening Linker-SEA segment for optimal biologic activity, and are functional when synthesized in either E. coli and insect cell systems.

General Significance

These results indicate that an Escherichiacoli-derived full-length His₈-tagged human MUC17 CRD1-L-CRD2 recombinant protein is a biologically active candidate for further development as a therapeutic agent.     

Mesenchymal stem cells: Molecular characteristics and clinical applications

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to differentiate into tissues of both mesenchymal and non-mesenchymal origin. MSCs can differentiate into osteoblastic, chondrogenic, and adipogenic lineages, although recent studies have demonstrated that MSCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages. Since their original isolation from the bone marrow, MSCs have been successfully harvested from many other tissues. Their ease of isolation and ex vivo expansion combined with their immunoprivileged nature has made these cells popular candidates for stem cell therapies. These cells have the potential to alter disease pathophysiology through many modalities including cytokine secretion, capacity to differentiate along various lineages, immune modulation and direct cell-cell interaction with diseased tissue. Here we first review basic features of MSC biology including MSC characteristics in culture, homing mechanisms, differentiation capabilities and immune modulation. We then highlight some in vivo and clinical evidence supporting the therapeutic roles of MSCs and their uses in orthopedic, autoimmune, and ischemic disorders.     

Multisite RNA binding and release of polypyrimidine tract binding protein during the regulation of c-src neural-specific splicing

We studied the role of polypyrimidine tract binding protein in repressing splicing of the c-src neuron-specific N1 exon. Immunodepletion/add-back experiments demonstrate that PTB is essential for splicing repression in HeLa extract. When splicing is repressed, PTB cross-links to intronic CUCUCU elements flanking the N1 exon. Mutation of the downstream CU elements causes dissociation of PTB from the intact upstream CU elements and allows splicing. Thus, PTB molecules bound to multiple elements cooperate to repress splicing. Interestingly, in neuronal WERI-1 cell extract where N1 is spliced, PTB also binds to the upstream CU elements but is dissociated in the presence of ATP. We conclude that splicing repression by PTB is modulated in different cells by a combination of cooperative binding and ATP-dependent dissociation.     

Characterization of GABAA receptor-mediated 36chloride uptake in rat brain synaptoneurosomes

gamma-Aminobutyric acid (GABA) receptor-mediated 36chloride (36Cl-) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated 36Cl- uptake in a concentration-dependent manner with the following order of potency: Muscimol greater than GABA greater than piperidine-4-sulfonic acid (P4S)greater than 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP) = 3-aminopropanesulfonic acid (3APS) much greater than taurine. Both P4S and 3APS behaved as partial agonists, while the GABAB agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated 36Cl- uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated 36Cl- uptake was also dependent on the anion present in the media. The muscimol response varied in media containing the following anions: Br- greater than Cl- greater than or equal to NO3- greater than I- greater than or equal to SCN- much greater than C3H5OO- greater than or equal to ClO4- greater than F-, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl- channel.