GPCR Compound Library

Some of the affected proteins have previously been associated with effects of prenatal protein undernutrition in mammalian models and will be discussed in more detail. The GLUT proteins are a family of facilitative transport proteins, catalyzing glucose uptake across the plasma membrane, the rate-limiting step in glucose metabolism. In mammals, GLUT1 is expressed ubiquitously and facilitates the basal glucose uptake, which is essential for growth and development in most cells. Expression of GLUT1 has previously been examined in other mammalian models of prenatal undernutrition, but no differences could be detected. Chickens exhibit a peculiar glucose transport and glucose homeostasis, since they are lacking GLUT4, the major insulin-responsive transporter. The mechanism for regulation of blood glucose concentration in chickens is not well understood. Furthermore, no information is available on the roles of the chickens GLUT isoforms in relation to glucose metabolism. Most likely, GLUT1 will act in maintaining basal glucose transport in most chicken cell types as in mammals, however the precise function of GLUT1 in the chicken remains to be elucidated. Chickens maintain an elevated level of blood glucose, which is supported by high rates of gluconeogenesis. In the present study, prenatal protein undernutrition caused an increase in PCK2 protein abundance, as opposite to the decreased FBP1 protein abundance and the decreased glycogen content. The liver glycogen content has previously been shown to change in a reciprocal way to the cytosolic PCK activity. Indeed, there are two forms of PCK found in most species, differing in their cellular localization: cytosolic PCK1 and mitochondrial PCK2. The relative abundance of both isoforms is dependent on the animal species and the growth stage of the animal. In the avian liver, the mitochondrial PCK activity is the most abundant one but during the perinatal period the cytosolic PCK activity increased considerable from a few days before hatching to 4 days after hatching. As PCK1 is the most abundant form in rats and has been linked previously with effects of prenatal protein undernutrition, the expression of both PCK1 and PCK2 was measured. Both genes were present in similar amounts in the liver at hatch, but were not influenced by the applied treatment. Rats and mice have been used extensively to examine the effects of the maternal diet on the programming of the progeny. In rat dams fed a protein-restricted diet an increase in PCK1 mRNA and increased activity was detected in liver of the progeny until 11 months of age, suggesting that programming of the metabolism also extends to the regulation of gene expression. A persistent increase in the gene expression of hepatic PCK, catalyzing the first, committed step of gluconeogenesis, leads to reduced ability of insulin to suppress hepatic glucose output.

TALE technology holds great promise in serving as a useful tool to decipher the functionality of genetic elements and in serving as a means to selectively switch on or off genes for therapeutic purpose. In the effort to selectively switch on gene expression using TALE technology, recent studies from two independent laboratories have demonstrated that robust and synergistic gene activation can be achieved using multiple TALE activators. Here we report that this synergistic effect could be further potentiated by up to 11-fold with a novel class of TALEbased activators, TBP-TALE. We demonstrated the potentiation capability of TBP-TALEs using two classical examples of silent, cell-type restricted genes, IL-2 and GM-CSF, in diverse cell lines at both the transcriptional and translational level. These unique TBP-based activators seem to function synergistically with the conventional VP64-TALEs on both genes and on a variety of cell lines that have been tested, indicating their universal potentiation activity in a diverse intracellular environment. The demonstrated potency of TBP-TALEs in synergizing with other VP64 activators to selectively switch on the expression of immunoregulatory genes such as IL-2 and GM-CSF has direct implications for targeted cancer immunotherapy and other similar applications. Although our studies, together with two recent publications, have clearly shown that multiple VP64-TALE activators alone or in combination with TBP-TALE can act synergistically to switch on silenced genes, the detailed mechanism of such a synergistic action has not been fully elucidated. One report has suggested that TALE activators can be designed with negligible regard for chromatin structure. However, based on extensive characterization of both the IL-2 and GM-CSF promoters, it is possible that their proximal and/or core promoter regions require extensive chromatin remodeling to activate gene expression. In supporting this notion, our data indicate that the robust TALE-mediated activation on the IL-2 promoter was in part due to altered chromatin accessibility possibly attributed to the action of VP64-TALE activators in collaboration with TBP-TALE directed initiation of transcription. As transcriptional initiation begins with the recruitment of TBP, it is plausible that targeted binding of TBP-TALE to the TATA box in cytokine gene promoters can bypass this rate limiting initial step of transcription for a silenced gene and facilitate mechanisms directed by VP64-TALEs such as, displacing or repositioning the nucleosome to allow for exclusive access of TALE activators to the promoter regulatory regions. In addition, other cooperative interactions between TBP and VP64-TALEs may also contribute to their synergistic activation on the cytokine genes. Potent transcriptional activators like VP16, in which our study uses four copies of it, interact with multiple components.

The sorbent cartridge could bind and retain IL-6 and/or p-cresol and its metabolites is consistent with the chemical-physical characteristics of its styrene resin that can establish strong non-covalent interactions with hydrophobic molecules and with albumin. A relevant question arises about the forms in which either pcresol or IL-6 bind to the resin. Both these molecules circulate, indeed, in the plasma both as protein-bound and as free forms. Specifically, p-Cresol and its derivatives are more than 90% bound to albumin whereas almost 70% of IL-6 circulates as a very high molecular complex with its soluble receptors. The filtering membrane of the convection stage of the HFR Supra apparatus has a low but measurable permeability to albumin with an albumin sieving coefficient of 0.02. Therefore, it lets an amount of albumin ranging around 1/40th of its plasma concentration cross the membrane and appear into the UF. This implies that uremic toxins, like p-cresol, can be transferred in the UF also in their protein bound forms. In addition, the free forms of p-cresol and of its metabolites are small enough to freely permeate through the convection stage filter. Therefore, both protein-bound and free p-cresol presumably passed through the dialysis membrane into the UF and become available for binding to the cartridge. Conversely, because of the very high molecular weight of its complex with soluble IL-6 receptors, it is likely that IL-6 passed into the UF only as a free molecule. This is consistent with the evidence that IL-6 concentration in the UF averaged 1/5th of the plasma concentration, the value expected for free IL-6 crossing through a membrane with IL-6 sieving coefficient of about 0.3. The evidence that we found low or undetectable IL-6 concentrations in the effluent from the cartridge suggests that the IL-6 that crossed the dialysis membrane of the first HFR stage to move into the UF was virtually all retained by the cartridge itself. In keeping with this result is the evidence that the ability of the UF incubation to trigger IL-6 gene expression and release in PBMC from healthy subjects was greatly reduced after its passage through the HFR cartridge sorbent bed. While the evidence discussed above suggests that HFR cartridge contributed to clear the plasma of our patients from uremic toxins, a role could have been played also by the second, diffusive stage of the HFR apparatus. As mentioned above, this portion of the HFR system operates as a conventional HD system in which diffusion represents the main mechanism of solute removal. However, the removal of p-cresol by this mechanism is expected to be small and that of IL-6 null. Indeed, less than 1% of total p-cresol circulates as free form whereas free IL-6 is still too large to cross the low flux membrane of the second HFR stage. As a whole these considerations suggest that a significant amount of p-cresol and all of IL-6 removal actually took place in the first HFR stage.

Moreover, protocorm-like bodies of Dendrobium orchid and Citrus madurensis embryonic axes required shorter PVS exposure times at 25uC than 0uC, i.e. 20 versus 60 min respectively. Whilst the time window for optimum PVS treatment is wider at lower temperatures, tropical species tend to respond better with warmer temperature treatment, e.g. Colocasia esculenta. The physical dimensions and permeability characteristics of the tissue under investigation profoundly affect the outcome of the cryoprotection and cryopreservation procedures. Smaller apices of garlic displayed higher regeneration after cryopreservation than large ones. Similarly, Nephelium ramboutan-ake shoot-tips of c. 2 mm tolerated cryopreservation well, as did 0.8 mm diameter axillary buds of Colocasia esculenta. The cryopreservation of mature zygotic embryos of recalcitrant seeds generally requires a reduction in tissue mass to facilitate cryoprotectant uptake. Usually, this involves the excision of the embryonic axis. In axes of recalcitrant seeds of sweet chestnut such surgical intervention results in a burst of superoxide, with further oxidative stress during subsequent desiccation. In this context, assuming that the protectants permeate sufficiently. Permeation of chemicals into the intercellular spaces and cells of plant tissues is compounded by many features. To enable the rapid permeation of the viability stain, triphenyl tetrazolium chloride, into oily tissues of pine seed, we previously used vacuum infiltration. Similarly, this system has been used to improve efficient gene transformation, the delivery of pathogenic bacteria into the intercellular spaces of plants to study pathogenplant cell interactions and the diffusion of an inhibitor of ethylene action so that pear fruits have prolonged storage. In addition, preliminary studies have shown that vacuum-assisted glycerol cryoprotectant infiltration can preserve the normal histology of rat leg muscle with no ice crystal formation after 3 weeks storage at 280uC. In this study, we developed and compared the efficacy of vacuum infiltration vitrification using PVS2 for the cryopreservation of seed embryos of three species with varying morphology, stress physiology and chemistry: Carica papaya ; Passiflora edulis ; and Laurus nobilis. These species have purported differences in seed storage characteristics. C. papaya has a high level of desiccation tolerance to about 5% moisture content, limited storability at 220uC, but tolerance of cryopreservation. P. edulis seeds may show reduced viability after drying to 5–6% moisture content, but the majority of dry seeds tolerate cryopreservation. Both species have spatulate embryos in copious endosperm. Finally, L. nobilis has seeds with a lowest safe moisture content of c. 24%, below which they are desiccation sensitive and successful moist storage at 0uC is limited to about 4 months.

Instead, we find that cytokine stimulation induces VCAM-1 expression by glioma cells, an observation of potential significance for understanding cytokine influences on glioma progression and dissemination. These experiments were prompted by efforts to use cytokinestimulation paradigms to increase IL13Ra2 expression on glioma cells and thereby increase the efficacy of IL13Ra2 targeted therapies for brain tumors. Based on the many studies which reported induction of IL13Ra2 on a variety of cell types following cytokine stimulation, we envisioned that this strategy for IL13Ra2 induction may be conserved for glioblastoma as well. Indeed, following cytokine stimulation, we did observe induction of a cell surface antigen on both primary glioblastoma cell lines and the monocyctic cell line THP-1, which was strongly detected by the commercially available putative IL13Ra2-targeted monoclonal antibody B-D13-PE. However, during the course of these studies, we encountered a series of discrepancies in the behavior of the B-D13-PE antibody that led us to question its binding specificity, and whether the induced antigen following cytokine stimulation was really IL13Ra2. In particular, following cytokine stimulation, B-D13-PE immunoreactivity did not correlate with either the immunoreactivity of the highlycharacterized IL13Ra2-specific goat polyclonal antibody AF146 or IL13Ra2 mRNA levels. Further, the cytokine induced B-D13-PE antigen did not bind IL-13 or elicit lysis by IL13Ra2-redirected CAR T cells, and B-D13-PE binding on cytokine stimulated cells could not be blocked by soluble IL13Ra2-Fc. Therefore, our data indicate that neither TNF/IL-4, TNF/IL-13, nor TNF alone induce cell surface IL13Ra2 upregulation on glioma cells, and therefore that these cytokine treatments are not a viable strategy for expanding the targetability of IL13Ra2 by immunotherapy. Instead, our data definitively demonstrate that the cytokine induced antigen recognized by B-D13-PE is VCAM-1, as demonstrated by B-D13 immunoprecipitation/mass spectrometry, as well as soluble receptor competition studies. Many studies have reported induction of IL13Ra2 on a variety of cell types following cytokine stimulation, and induction of IL13Ra2 has been reported to be involved in TGF-b1 production. However, all of these studies used the B-D13 antibody to evaluate protein induction, and thus may have inadvertently mis-identified the induction of IL13Ra2 protein following cytokine stimulation. It should be noted, however, that qPCR and knockdown studies do support IL13Ra2 induction in some cell types. In fact, consistent with previous reports, we find that THP-1 cells show induction of IL13Ra2 mRNA 13 to 15-fold after overnight treatment with TNF and IL-13 or IL-4, although the level of IL13Ra2 expression was more than 13.6-fold lower than that expressed by the U251T glioma cell line and not at sufficient levels to be detected by flow cytometry using the IL13.