For this, we determined if DMOG can protect MEFs lacking both the Suv39h1 and Suv39h2 methyltransferases. The ability of DMOG and CoCl2 to increase Hif1a levels was not altered in the SuvDKO MEFs. Further, whereas DMOG functioned as a radioprotector in normal MEFs, the protective effect of DMOG was significantly reduced in the SuvDKO MEFs. This indicates that the reduced ability of DMOG to protect SuvDKO MEFs may be attributed to both the loss of Hif1a dependent increase in the Suv39h1 methyltransferase, as well as changes in other Hif1a target genes. Overall, figure 5 demonstrates that DMOG can protect cells form radiation by stabilizing the Hif1a transcription factor, and increasing expression of genes, including the Suv39h1 methyltransferase, which can promote cell survival. Finally, we sought to determine if DMOG could protect cells from DNA damage at the level of the whole organism. Hif1a activation can transcriptionally activate a wide range of genes, including VEGF, which promote angiogenesis, and erythropoietin, which mobilizes bone marrow and improves blood parameters. It is likely that these factors may be important in promoting recovery of sensitive tissues, such as the bone marrow and gastrointestinal tract, from total body irradiation. In order to assess if the radioprotective effects of DMOG can be detected at the level of the whole organism, we examined if DMOG had protective effects in a murine total body irradiationmodel. DMOG was injected ip into either C57BL/6J or Balb/c mice, two widely used murine TBI model systems. DMOG alone did not cause any toxicity, with 100% of the mice surviving at 30 days, in agreement with previous studies using DMOG. TBI at 8Gy caused 80% and 100% lethality in saline-treated C57BL/6J and Balb/c mice respectively. However, DMOG treatment significantly improved the survival of both C57BL/6J and Balb/c mice and rescued them from radiation induced lethality. Activation of Hif1a by DMOG results in the transcriptional PR-171 upregulation of many genes and growth factors and is associated with increased cell survival in culture and protection of mice from whole body irradiation. These results clearly demonstrated that DMOG exhibits radioprotective potential in murine models. The Hif1a transcriptional regulatory protein controls the expression of genes which promote cell survival under conditions of low oxygen tension. Hif1a can switch cell metabolism towards increased expression of growth factors and anti-apoptotic factors as well as switching of metabolic pathways, allow the cell to maintain energy levels under hypoxic conditions. Hif1a levels are controlled through direct hydroxylation of Hif1a by the PHD2 prolylhydroxylase, leading to degradation of Hif1a under normal oxygen tension. Small molecule inhibitors of prolylhydroxylases have been developed which inhibit PHD2 and related enzymes, leading to stabilization of Hif1a and upregulation of the hypoxia response. Here, we have shown that stabilization of Hif1a using the prolylhydroxylase inhibitor DMOG protects both MEFs and mice from the cytotoxic effects of exposure to IR. This is consistent with previous studies high throughput screening demonstrating that tumor cells containing constitutively high levels of Hif1a are more resistant to both chemotherapy and radiotherapy. Increasing Hif1a levels in normal cells with prolylhydroxylase inhibitors such as DMOG therefore represents a novel pathway for the development of new and effective radioprotective agents. Our results clearly show that DMOG requires Hif1a to protect cells from radiation, since suppression of Hif1a with shRNA abolished the protective effect of DMOG.

Interestingly, we recently showed that only AF4NMLL but not the reciprocal Cycloheximide cost translocation product, MLLNAF4, lacking the Taspase1 cleavage site, can cause proB ALL in a murine model. Thus, proteolytic cleavage of MLL-fusion proteins by Taspase1 is considered a critical step for MLL-mediated tumorigenesis, although the molecular details are not yet resolved. Besides Taspase1’s role in leukemogenesis the protease was suggested to be also overexpressed solid tumors. In this respect, recent data indicate that also other regulatory proteins, such as the precursor of the Transcription Factor IIAor Drosophila HCF, are Taspase1 targets. Hence, there is an increasing interest in defining novel Taspase1 targets. However, the molecular mechanisms how Taspase1 affects biological functions through site-specific proteolysis of its substrates and what other cellular programs are regulated by Taspase1’s degradome under normal or pathophysiological conditions is completely unknown. Besides genetic instruments, chemical decoys allowing the targeted inhibition/activation of proteins are powerful tools to dissect complex biological pathways. Small molecules that allow a chemical knock out of a cellular reaction or a cell phenotype can be selected by phenotypic screens, and used as molecular tools to identify previously uncharacterized proteins and/or molecular mechanisms. Hence, chemogenomics as studying the interaction of biological systems with exogenous small molecules, i.e., analyzing the intersection of biological and chemical spaces, seems an attractive approach to also dissect Taspase1 functions. Unfortunately, Taspase1��s catalytic activity is not affected by common protease CHIR-99021 GSK-3 inhibitor inhibitors and no small molecule inhibitors for this enzyme are currently available to dissect Taspase1’s function in vivo. As biochemical data or potential drugs must be effective at the cellular level, reliable cell-based assaysfor Taspase1 are urgently needed. Often, redistribution approaches, as cell-based assay technology that uses protein translocation as the primary readout have been used to study the activity of cellular signaling pathways. Protein targets are labeled with autofluorescent proteins and are read using high-throughput, microscope-based instruments. Although, protein translocation assays have the potential for high-content, high-throughput screeningapplications, such assays are generally not used for proteases. Here, the spatial and functional division into the nucleus and the cytoplasm was exploited to design a translocation-based Taspase1-biosensor assay. The CBA was adapted on a HTS platform, employed to identify potential Taspase1 small molecule inhibitors, and was used to study Taspase1 function in living cells. The robust performance of the TS-Cl2+CBA met critical requirements for high content screening: the biosensor was nontoxic, localized to the cytoplasm in the absence of ectopically expressed Taspase1, and efficiently accumulated in the nucleus following Taspase1-specific cleavage. Hence, we tested whether the assay can also be used on a high-throughput microscopy based screening platform. As cell lines inducibly expressing biosensors may facilitate certain HCS/HTS applications, we generated stable Tet-off TSCl2 + TRE cell lines. The tetracycline -regulated system has been used successfully in various applications. Whereas expression of TS-Cl2 + TRE was blocked in the presence of doxycycline, Dox removal induced TS-Cl2+ TRE expression. Cleavage of TS-Cl2+TRE by the endogenous Taspase1 subsequently resulted in nuclear accumulation of the biosensor.

However, in contrast to other experiments performed during the course of this study, the difference between the 24 hours post-infection treatment schedule and the control group did not quite reach significance. Intrigued by this finding, we conducted a separate experiment in which we determined the effect of intranasal iota-carrageenan treatment on viral titer of NSC 136476 500579-04-4 infected mice. We infected 5 mice per group as before and either started intranasal therapy with iotacarrageenan or oral therapy with Z-VAD-FMK oseltamivir 24 and 48 hours post infection as before, respectively. Subsequently, groups of mice were sacrificed 48 or 120 hours post infection and after semi-daily therapy and viral titers were determined from pooled specimens derived from the nasal cavity and lung by plaque assays. As shown in Figure 6B, intranasal treatment of mice with iota-carrageenan results in an immediate reduction of viral particles in the nasal cavity 2 days and even more pronounced 5 days post infection, in the same order of magnitude as the neuraminidase inhibitor oseltamivir. Conversely, while we could not determine a titer reduction in the lung 48 hours post infectionin the iota-carrageenan-treated group, we could clearly show a strong reduction of viral particles in the lungs of iota-carrageenan-treated mice 5 days post infection as compared to the control group. Importantly, iotacarrageenan treatment seemed to be as efficient as an oseltamivir therapy and as before, we could see a benefit with respect of viral particle reduction in the nose and lung even if therapy was started as late as 2 days post infection. Intranasal therapy of infected mice with iota-carrageenan results in a survival benefit for mice and seems to be a direct consequence of a reduction in viral particles present in the nose and consequently in the lung at later time points of the infection, respectively. In this report we demonstrate that iota-carrageenan, a biopolymer derived from red seaweed, is a potent inhibitor of influenza virus infectivity in vitro and in vivo. The report describes cell culture studies, demonstrates the antiviral activity of iotacarrageenan in mouse influenza infection models and proposes a mode of action. The antiviral activity of iota-carrageenan against several virus types other than influenza has been studied more than 20 years ago. Antiviral activity was found against herpes simplex virus type1 and 2 at an IC50 of 2 and 10 mg/ml, respectively. In the same report, iota-carrageenan was found ineffective against measles virus, adenovirus type 5, poliovirus and vesicular stomatitis virus. Our results indicate that iota-carrageenan is active against influenza A viruses at ten times lower concentrations when compared with HSV-1 in a standard plaque reduction assay. This is comparable to our in vitro data of human rhinoviruses, but does not reach the low effectivity dosage range that has been described for papillomaviruses. Both iotaand kappa-carrageenan protected MDCK cells from virusinduced cell death at an MOI of 0.01in a dosedependent manner. Moreover, maintenance of MDCK cells in the presence of iota-carrageenan up to 96 hours post infection with H1N1 also resulted in a dramatic reduction of viral titers by 2-4 logs, indicative of a protective effect of iota-carrageenan with regard to the spread and release of viral particles from previously infected MDCK cells. However, an increased amount of input virus gradually reduces the protective effect. Therefore, we conclude that the antiviral effect of carrageenan is dependent on the relative amount of input virus in both cases.

Infections associated with methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium have resulted in increasing nosocomial health concerns for both patients and medical professionals. Thus, there is an urgent need for new antibacterial agents with innovative mechanisms of action. Filamenting temperature-sensitive mutant Z, an analogue of eukaryotic tubulin, is an essential and highly conserved bacterial cytokinesis protein. GSK1363089 During bacterial cell division, FtsZ monomers self-assemble into a Z-ring, a highly dynamic cytoskeleton scaffold generated at the site of septum formation. The mechanism regulating assembly and organization of FtsZ into a ring-like structure involves GTP binding and hydrolysis, modulated by the interaction of the N-terminal nucleotide binding domain of one FtsZ monomer with the C-terminal GTPase activating domain on the adjacent FtsZ monomer. Subsequently, FtsZ recruits other proteins to form a cell-division complex known as the divisome. Once the divisome is fully assembled, bacterial cell division is achieved by coordinated constriction and splitting of the daughter cells. Everolimus recent studies suggest that inhibition of bacterial cell division proteins with an essential role in bacterial cytokinesis, such as FtsZ, is a promising approach against antibiotic-resistant bacterial infections. A number of small molecule inhibitors of FtsZ have already been shown to prevent FtsZ polymerization and inhibit bacterial cell division. The molecules bind to one of two alternative sites of FtsZ : at the N-terminal GTP binding site, or at the C-terminal interdomain cleft. Compounds targeting the highly conserved GTP binding site mimic the natural substrate of the enzyme and might have potential advantages for developing broad-spectrum antibacterial agents. However, because GTP binding sites are present in a number of human proteins, GTP-mimetic compounds might have potential liabilities related to the off-target-associated activity. Thus, the C-terminal interdomain cleft formed by residues from the C-terminal b-sheet, T7-loop and H7-helix, offers an alternative opportunity for the design of FtsZ inhibitors with therapeutic potential in antibiotic-resistant bacterial diseases. Berberine is a plant alkaloid with a long history of medicinal use in traditional Chinese and native American medicines. Berberine extracts show significant antimicrobial activity against bacteria, viruses and fungi. Its potential mechanisms of antimicrobial activity include the suppression of cell adhesion and migration, and inhibition of microbial enzymes. Moreover, recent literature reports demonstrated that berberine is active against Gram-positive bacteria with minimum inhibitory concentration values in the range of 100�C 400 mg/mL by targeting the cell division protein FtsZ. Therefore, berberine is an attractive lead for the development of potent FtsZ inhibitors. Given the availability of X-ray crystal structures of FtsZ, molecular docking is particularly appealing for guiding the chemical derivatization of berberine. Previous studies suggested that berberine binds FtsZ in a hydrophobic pocket. In this paper we report the design and biological study of a series of 9-phenoxyalkyl berberine derivatives with potent inhibition of FtsZ GTPase activity and broadspectrum of antibacterial activity. Natural products and semi-synthetic derivatives provide a rich source of bioactive compounds for the development of new antibacterial agents. However, in the past decade most of the new chemical entities that reached the clinical practice were derived from the same natural scaffolds. Berberine has been traditionally used to treat microbial infections. At the doses commonly used, the compound is considered safe.

We have recently demonstrated that C3bot1E174Q selectively delivers proteins and enzymes into cultured macrophages including primary human macrophages derived from monocytes from blood donors. Because C3-based transporters target monocytes/macrophages in general, they would not serve for a selective drug delivery into osteoclasts after a systemic application. However, a targeted local application of either wildtype C3 for Rho-inhibition in osteoclasts or C3-derived transport systems for targeted drug delivery into osteoclasts might be an appropriate approach to manipulate osteoclastogenesis and/or osteoclast functions, e.g. to improve the osseous integration of orthopaedic implants by suppressing osteoclast activity at the implant surface. Local application in bone and controlled release of C3 proteins or C3transporters from orthopaedic implant surfaces could be achieved by the use of biocompatible carriers such as resorbable polymers or hydrogels. Matrix stiffness is an important regulator of cell behavior. Stiffness has been shown to affect cell morphology and spreading, proliferation, migration, apoptosis rate, and differentiation. However, most cell studies are performed on tissue culture plastic, which largely fails to replicate the mechanics and microenvironment that cells experience in vivo. Tissue culture plastic is commonly cited as having an elastic modulus of approximately 1 GPa, whereas tissues in the body are less than 100 kPa, with brain having an elasticity less than 1 kPa, muscle around 10 kPa, and bone around 100 kPa. The effects of matrix stiffness are typically evaluated by analyzing cell behavior in different gel systems. Stiffness or elasticity can be varied by simply changing the crosslinking Rapamycin density. Several different hydrogel systems have been investigated including polyacrylamide gels, alginate, collagen, matrigel, chitosan, and hyaluronic acid. Because BMS-907351 c-Met inhibitor substrate stiffness regulates so many cellular functions, we wanted to investigate its role in the uptake of cell-penetrating peptides. Although the exact mechanism of cell-penetrating peptide uptake is still debated, investigators generally agree that uptake occurs via one or more of the endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, or through membrane destabilization or formation of transient pores. Our lab has designed and reported on a family of peptide inhibitors of mitogen kinase activated protein kinase- activated protein kinase 2, a kinase important in regulating inflammation through the regulation of proinflammatory cytokines. These inhibitors consist of a cell-penetrating peptide domain for intracellular delivery and a therapeutic domain that inhibits MK2. Recently, we demonstrated that the peptide variant YARAAARQARAKALARQLGVAA was taken up primarily through caveolae-mediated endocytosis in mesothelial cells. However, when comparing between data obtained from in vitro cell and in vivo animal models, we observed an unusual effect: concentrations of the YARA MK2 inhibitor peptide required for efficacy in cells ranged from 1000�C3000 mM; however, the concentration required for efficacy in animal models was ten to one hundred-fold less, in the range of 10�C100 mM. This phenomenon opposes what is normally observed in the pharmaceutical industry, as drug concentrations must usually increase to demonstrate efficacy when moving from cell culture to animal models due to metabolism and non-uniform distribution within the body. We hypothesized that the discrepancy observed in peptide concentration required to achieve efficacy in studies in vitro as compared to studies in vivo was due to the unrealistic stiffness of tissue culture polystyrene. Using a technique pioneered by Pelham and Wang and refined by others, the role of substrate stiffness.