Micheliolide Derivative DMAMCL Inhibits Glioma Cell Growth In Vitro and In Vivo

BackgroundThere is no highly effective chemotherapy for malignant gliomas to date. We found that dimethylaminomicheliolide (DMAMCL), a selective inhibitor of acute myeloid leukemia (AML) stem/progenitor cells, inhibited the growth of glioma cells.MethodsThe distribution of DMAMCL in brain was analyzed by an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS) system. The anti-tumor evaluations of DMAMCL in vitro were performed by MTT, FACS and RT-PCR. In vivo, the mixture of C6 cells and matrigel was injected into caudatum, and the anti-tumor activity of DMAMCL was evaluated by tumor growth and rat survival. The toxicity of DMAMCL was evaluated by body weight, daily food intake, hematological or serum biochemical analyses, and histological appearance of tissues.ResultsThe IC₅₀ values of DMAMCL against the C6 and U-87MG cell lines in vitro were 27.18 ± 1.89 μM and 20.58 ± 1.61 μM, respectively. DAMMCL down-regulated the anti-apoptosis gene Bcl-2 and increased apoptosis in C6 and U-87MG cells in a dose-dependent manner. In a C6 rat tumor model, daily administration of DMAMCL for 21 days reduced the burden of C6 tumors by 60% to 88% compared to controls, and more than doubled the mean lifespan of tumor-bearing rats. Distribution analysis showed that the DMAMCL concentration was higher in the brain than in plasma. Evaluations for toxicity revealed that oral administration of DMAMCL at 200 or 300 mg/kg once a day for 21 days did not result in toxicity.ConclusionsThese results suggest that DMAMCL is highly promising for the treatment of glioma.     

Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo

N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), which transfer an acetyl group from acetyl coenzyme A to the alpha amino group of a nascent polypeptide. Nt-acetylation has emerged as an important protein modifier, steering protein degradation, protein complex formation and protein localization. Very recently, it was reported that some human proteins could carry a propionyl group at their N-terminus. Here, we investigated the generality of N-terminal propionylation by analyzing its proteome-wide occurrence in yeast and we identified 10 unique in vivo Nt-propionylated N-termini. Furthermore, by performing differential N-terminome analysis of a control yeast strain (yNatA), a yeast NatA deletion strain (yNatAΔ) or a yeast NatA deletion strain expressing human NatA (hNatA), we were able to demonstrate that in vivo Nt-propionylation of several proteins, displaying a NatA type substrate specificity profile, depended on the presence of either yeast or human NatA. Furthermore, in vitro Nt-propionylation assays using synthetic peptides, propionyl coenzyme A, and either purified human NATs or immunoprecipitated human NatA, clearly demonstrated that NATs are Nt-propionyltransferases (NPTs) per se. We here demonstrate for the first time that Nt-propionylation can occur in yeast and thus is an evolutionarily conserved process, and that the NATs are multifunctional enzymes acting as NPTs in vivo and in vitro, in addition to their main role as NATs, and their potential function as lysine acetyltransferases (KATs) and noncatalytic regulators.Modifications greatly increases a cell''s proteome diversity confined by the natural amino acids. As more than 80% of human proteins, more than 70% of plant and fly proteins and more than 60% of yeast proteins are N-terminally acetylated (Nt-acetylated),¹ this modification represents one of the most common protein modifications in eukaryotes (1–5). Recent studies have pointed to distinct functional consequences of Nt-acetylation (6): creating degradation signals recognized by a ubiquitin ligase of a new branch of the N-end rule pathway (7), preventing translocation across the endoplasmic reticulum membrane (8), and mediating protein complex formation (9). Nt-acetylation further appears to be essential for life in higher eukaryotes; for instance, a mutation in the major human N-terminal acetyltransferase (NAT), hNatA, was recently shown to be the cause of Ogden syndrome by which male infants are underdeveloped and die at infancy (10). Unlike lysine acetylation, Nt-acetylation is considered an irreversible process, and further, to mainly occur on the ribosome during protein synthesis (11–15). In yeast and humans, three NAT complexes are responsible for the majority of Nt-acetylation; NatA, NatB and NatC, each of which has a defined substrate specificity (16). NatA acetylates Ser-, Ala-, Gly-, Thr-, Val- and Cys- N-termini generated on removal of the initiator methionine (iMet) (1, 17–19). NatB and NatC acetylate N-termini in which the iMet is followed by an acidic (20–23) or a hydrophobic residue respectively (24–26). Naa40p/NatD was shown to acetylate the Ser-starting N-termini of histones H2A and H4 (27, 28). NatE, composed of the catalytic Naa50p (Nat5p) has substrate specificity toward iMet succeeded by a hydrophobic amino acid (29, 30). As largely the same Nt-acetylation patterns are found in yeast and humans, it was believed that the NAT-machineries were conserved in general (31). However, the recently discovered higher eukaryotic specific NAT, Naa60p/NatF, was found to display a partially distinct substrate specificity in part explaining the higher degree of Nt-acetylation in higher versus lower eukaryotes (4).Human NatA is composed of two main subunits: the catalytic subunit hNaa10p and the auxiliary subunit, hNaa15p that is presumably responsible for anchoring the complex to the ribosome (14, 19). The chaperone-like HYPK protein is also stably associated with the NatA subunits and may be essential for efficient NatA activity (32). In addition, hNaa50p was shown to be physically associated with hNatA, however it is believed not to affect NatA activity (14, 33, 34). hNaa50p was also shown to exhibit N^ε-acetyltransferase (KAT) activity (29), however, the structure of hNaa50p with its peptide substrate bound strongly indicates that the peptide binding pocket is specifically suited to accommodate N-terminal peptides, as opposed to lysine residues (35). The human NatA subunits are associated with ribosomes, but interestingly, significant fractions are also nonribosomal (19, 30, 32). Of further notice, the catalytic subunits, hNaa10p and hNaa50p, were also found to partially act independently of the hNatA complex (30, 36).Recent studies have identified novel in vivo acyl modifications of proteins. Mass spectrometry data of affinity-enriched acetyllysine-containing peptides from HeLa cells showed the presence of propionylated and butyrylated lysines in histone H4 peptides (37). Similar analyses also showed the presence of propionylated lysines in p53, p300 and CREB-binding protein (38) besides the yeast histones H2B, H3 and H4 (39). Propionylated or butyrylated residues differ by only one or two extra methyl moieties as compared with their acetylated counterparts, thereby adding more hydrophobicity and bulkiness to the affected residue. To date, no distinct propionyl- or butyryltransferases responsible for these modifications have been identified. However, by using propionyl coenzyme A (Prop-CoA) or butyryl coenzyme A (But-CoA) as donors in the enzyme reaction, it was shown that some of the previously characterized lysine acetyltransferases (KATs) are able to respectively catalyze propionylation and butyrylation of lysine residues both in vitro (37, 40–42) and in vivo (38, 41). Similarly, it has been shown that lysine deacetylases also are capable of catalyzing depropionylation (40, 41, 43, 44) and debutyrylation (44) (see review (45)).Interestingly, mass spectrometry data also suggested that propionylated N-termini are present in human cell lines (46, 47). Until today, an N-terminal propionyl transferase (NPT) catalyzing N-terminal propionylation (Nt-propionylation) has to our knowledge not been identified.In this study, we hypothesized that NATs might have the ability to act as NPTs. In vitro experiments using purified hNaa10p, hNaa50p or immunoprecipitated human NatA complex indeed confirmed their intrinsic capacity to catalyze Nt-propionylation toward synthetic peptides. NatA was also found capable of Nt-butyrylation in vitro. By means of N-terminomics, we further investigated the presence of yeast Nt-propionylated proteins in vivo. Indeed, we found evidence for Nt-propionylation being a naturally occurring modification in yeast. Interestingly, in a yeast strain lacking NatA, we observed a loss in Nt-propionylation and Nt-acetylation for several NatA substrates, as compared with a control yeast strain expressing endogenous NatA or a strain ectopically expressing hNatA. Thus, besides acting as NATs, yeast and human NatA can act as NPTs and we thus demonstrate for the first time that NATs have the capacity of both acetylating and propionylating protein N-termini in vivo and in vitro.     

Pseudocompact-Type Growth and Conversion of Growth Types of Strains of Staphylococcus aureus In Vitro and In Vivo

Some strains of Staphylococcus aureus grew as compact colonies in Brain Heart Infusion-serum-soft agar but as diffuse colonies in a modified Staphylococcus 110-serum-soft agar. These strains were designated "pseudocompact." Strains showing compact-type colonial morphology in both media were designated "compact," whereas strains showing diffuse-type growth in both media were designated "diffuse." It was observed that the most recently isolated strains of S. aureus were of the pseudocompact type, whereas most stock culture strains tested were of the compact type. Using cultures recently isolated from clinical material, it was shown that pseudocompact strains convert to compact-type growth after prolonged incubation. Interconversion of compact, diffuse, and pseudocompact growth forms could be induced in vitro by appropriate cultural conditions, and conversion of growth type was also observed in vivo. Femoral abscesses produced in mice by four different compact-type strains showed conversion to diffuse or pseudocompact-type growth during the course of the infection.     

CRMP-2 Is Involved in Axon Growth Inhibition Induced by RGMa In Vitro and In Vivo

Repulsive guidance molecule-a (RGMa) is associated with axon growth inhibition in different central nervous system (CNS) injuries, but its signaling pathways remain unclear. We examined the involvement of collapsin response mediator protein-2 (CRMP-2), a common downstream target of Rho-kinase and GSK-3β, in vitro by culturing neonatal rat primary cortical neurons with RGMa protein, Rho-kinase inhibitor (Y-27632), and GSK-3β inhibitor. We examined CRMP-2 in vivo by suppressing RGMa expression using recombinant adenovirus (rAd-shRGMa) in a rat MCAO/reperfusion model. RGMa induced neurite retraction and CRMP-2 phosphorylation in vitro, which were reversed by either Rho-kinase or GSK-3β inhibitors. After MCAO/reperfusion in rats, pCRMP-2 protein was greatly increased in the ischemic cortex, axons were damaged severely, Neurofilament-200 (NF-200) expression was significantly decreased, and neurological deficits were significant, which were all improved by down-regulating RGMa. We concluded RGMa inhibits axon growth by phosphorylating CRMP-2 via both Rho-kinase and GSK-3β signaling pathways.     

In Vitro and In Vivo Characterization of Pyocin

Pyocin, a bacteriocin obtained from lysates of ultraviolet-induced cultures of Pseudomonas aeruginosa was characterized in vitro and in vivo after 1,000-fold purification by chemical, column, and differential centrifugation procedures. Electron micrographs of negatively stained pyocin preparations contained rod-shaped particles which resembled the contractile tail protein of the T-even phages of Escherichia coli. Although two separate and distinct pyocin fractions were eluted from diethylaminoethyl cellulose (pH 7.5) during the purification procedure, the particles appeared identical. In addition, the two fractions exhibited a close correlation between their titers and the particle numbers as observed in the electron microscope. The particles were approximately 20 by 90 mmu with a core diameter of 5 mmu and a sheath length of 50 mmu. Neither intact phage nor ghosts were seen in any of the preparations, although ringlets of two different diameters, which appeared to correspond to the diameters of the sheath and inner core, were observed. Other studies indicated that, although crude preparations were stable to freezing and thawing, purified preparations lost all of their activity under similar treatment. However, the addition of 50% glycerol to purified preparations completely protected activity. Conversely, aged normal human or rabbit sera enhanced the antibacterial activity of pyocin approximately fourfold, although serum albumin and hemoglobin had no effect. In vivo studies indicated that purified pyocin was not lethal for mice when injected intraperitoneally in concentrations of 28,000 to 1,400,000 units (5.6 to 276 mug of protein), nor was 7,200 to 36,000 units dermonecrotic for rabbits.     

The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.     

In Vivo,In Vitro,and In Silico Characterization of Peptoids as Antimicrobial Agents

Bacterial resistance to conventional antibiotics is a global threat that has spurred the development of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N-substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicrobial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demonstrate the promising therapeutic potential of peptoids as antimicrobial agents.     

Regulation of Pyruvate Decarboxylase In Vitro and In Vivo

Results presented in this paper strongly support the view thatregulation of the key enzyme of alcoholic fermentation, pyruvatedecarboxylase (PDC), is achieved in a number of ways, all associatedwith possible lowering of the cytoplasmic pH during anoxia.These mechanisms include not only the well-known acid pH optimumof PDC, but also long-term, reversible changes in characteristicsof the enzyme established both in vitro and in vivo. Following transfer of desalted extracts from pH 6.0 to 7.4,maximal activity of PDC was decreased, while there was a considerableincrease in the lag before maximal activity was reached. Similarchanges in enzyme characteristics were observed when wheat (Triticumaestivum L. cv. Gamenya) roots and rice (Oryza sativa L. cv.Calrose) coleoptiles were transferred from anoxic to aerobicsolutions, provided PDC was assayed within 10 min of the startof maceration. All of the above changes were usually readilyreversible when extracts were returned to pH 6.0, or when plantswere returned to anoxic solutions. Additional regulation of PDC would be achieved by the S_0.5 forpyruvate which is 0.75 mol m^–3 at pH 6.0, 1.0 mol m^–3at pH 6.8, and 2.5 mol m^–3 at pH 7.4; the latter is wellabove estimates for pyruvate concentrations in the cytoplasmof aerated tissues. We assess that the combined effects of the acid pH optimum,the high S_0.5 at pH 7.4 and the long-term decreases in activityobserved during incubation at pH 7.4 would reduce PDC activityin aerobic cells to at most 7% of the activity in anoxic cells.Possible additional controls for the pathway of alcoholic fermentationare briefly considered. Key words: PDC, regulation, anoxia     

Antithrombotic Activities of Luteolin In Vitro and In Vivo

The objectives of present study were to investigate whether luteolin affects procoagulant proteinase activity and fibrin clot formation and influences thrombosis and coagulation in Sprague–Dawle rats. Luteolin significantly inhibited the enzymatic activity of thrombin and FXa activity by 29.1% and 16.2%. Luteolin also inhibited fibrin polymer formation in turbidity and microscopic analysis using fluorescent conjugate. Coagulation assay of luteolin was found to prolong activated partial thromboplastin time and prothrombin time. Moreover, luteolin protected the development of oxidative stress induced thrombosis in the FeCl₃‐induced carotid arterial thrombus model. This study demonstrated that luteolin may be useful by reducing or preventing thrombotic challenge and can help us better understand the antithrombotic action of luteolin.     

Fusion Toxin BLyS-Gelonin Inhibits Growth of Malignant Human B Cell Lines In Vitro and In Vivo

B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. The biological activity of BLyS is mediated by three cell surface receptors: BR3/BAFF-R, TACI and BCMA. The expression of these receptors is highly restricted to B cells, both normal and malignant. A BLyS-gelonin fusion toxin (BLyS-gel) was generated consisting of the recombinant plant-derived toxin gelonin fused to the N-terminus of BLyS and tested against a large and diverse panel of B-NHL cell lines. Interestingly, B-NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursor-acute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with low picomolar EC₅₀ values. BLyS receptor expression did not guarantee sensitivity to BLyS-gel, even though the construct was internalized by both sensitive and resistant cells. Resistance to BLyS-gel could be overcome by treatment with the endosomotropic drug chloroquine, suggesting BLyS-gel may become trapped within endosomal/lysosomal compartments in resistant cells. BLyS-gel induced cell death was caspase-independent and shown to be at least partially mediated by the “ribotoxic stress response.” This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment was shown to localize to sites of disease, rapidly reduce tumor burden, and significantly prolong survival in xenograft mouse models of disseminated BCP-ALL, DLBCL, and MCL. Together, these findings suggest BLyS has significant potential as a targeting ligand for the delivery of cytotoxic “payloads” to malignant B cells.     

Methyl Gallate from Galla rhois Successfully Controls Clinical Isolates of Salmonella Infection in Both In Vitro and In Vivo Systems

Galla rhois is a commonly used traditional medicine for the treatment of pathogenic bacteria in Korea as well as in other parts of Asia. Methyl gallate (MG), a major component of Galla Rhois, exhibits strong antibacterial activity, but its mechanism of action against Salmonella spp. is unclear. In the present study, we investigated the antibacterial actions of MG against Salmonella. The antibacterial activity determined by broth dilution method indicated that the antibacterial activity of MG against Salmonella strains ranged from 3.9 to 125 µg/ml. In vitro bacterial viability test indicated that MG significantly decreased the viability of Salmonella over 40% when combined with ATPase inhibitors. The time-kill curves showed that a combined MG and ATPase inhibitors (DCCD and NaN3) treatment reduced the bacterial counts dramatically after 24 h. Oral administration of MG showed a strong anti-bacterial activity against WS-5 infected BALB/c mice. In contrast to the untreated Salmonella infected control animals, MG treated groups showed no clinical symptoms of the disease, such as lethargy and liver damage. It was observed that MG treatment significantly increased the survival of animals from Salmonella infection, while in untreated groups all animal succumbed to disease by the sixth day post infection. Thus, the present study demonstrates the therapeutic ability of MG against Salmonella infections.     

Effects of Actinobolin on Growth and Some Metabolic Activities of Cariogenic Streptococci In Vitro and In Vivo

Actinobolin, a known inhibitor of protein synthesis, has been shown not to interfere selectively with acid production or dextransucrase activity in a cariogenic streptococcus when the antibiotic is added to a concentration of 500 mug/ml. It has also been shown that actinobolin does not alter the total in vivo flora of the oral cavity of the rat when tested in a rat caries model system. A culture of cariogenic streptococci, adapted to in vitro growth in the presence of 1 mg of actinobolin per ml, has also been isolated.