Discovery of dual Axl/VEGF-R2 inhibitors as potential anti-angiogenic and anti-metastatic drugs for cancer chemotherapy

Axl tyrosine kinase has been shown to be involved in multiple pathways contributing to tumor development, angiogenesis, and metastasis. High Axl expression has been observed in many human tumors where it appears to confer aggressive tumor behavior. Here we present several series of dual Axl-VEGF-R2 kinase inhibitors based on extensive optimization of an acyl diaminotriazole. It was hypothesized that dual inhibition of these two receptor tyrosine kinases may have a synergistic affect in inhibiting tumor angiogenesis and metastasis. One of these molecules, R916562 showed comparable activity to Sunitinib in two mouse tumor xenograft models and a mouse corneal micropocket model.     

Temporal variation and succession in an algal-dominated high intertidal assemblage

We determined whether temporal variation and succession were similar among sites with similar species composition by sampling unmanipulated and cleared plots in a high intertidal assemblage dominated by Endocladia muricata and Mastocarpus papillatus. Sampling was done for 6 years at six sites spanning over 4° of latitude in California. Ten 1×2-m permanent plots were chosen in the central portion of the assemblage at each site. Four of these served as unmanipulated controls, three were cleared (scraped and burned) in the spring of 1985, and three were cleared in the fall of 1985. The cover of sessile and density of motile species were determined by subsampling within the plots from 1985 until 1991. Recovery of the clearings was determined by their similarity to the controls. The algae E. muricata, M. papillatus, and Fucus gardneri, and the barnacle Balanus glandula, were the most abundant sessile organisms in the control plots, although the latter never exceeded 12% cover at any site. The grazing gastropods Littorina scutulata/plena, various limpets, and Tegula funebralis were the most common mobile organisms. The species composition of the common species remained constant in the control plots over the study period and there were few large changes in relative abundance. Significant seasonal variation was detected in 11 species but variation was commonly site-specific.Ephemeral algae were abundant during early succession at only two of the six sites, and barnacle cover was low (<15% cover) at four sites and moderate (15-50% cover) at the remaining two throughout succession. Recovery rate varied considerably among sites and between times of clearing (1-10%/month). Correlations between ephemeral algae and grazer abundance, and between these variables and recovery rate were not significant. The effects of grazers on recovery rate were only evident at one site where they appeared to reduce an initially high cover of ephemeral algae and delay the establishment of perennials. Some of the largest differences in recovery rate were between clearing times, associated with differences in the phenology of the dominant perennial algae. In spite of these differences, most plots recovered by the end of the study period.These results indicate that the assemblages in the control plots at each site were relatively stable and, while successional pathways and processes varied, the assemblage at most sites still recovered. Current models, based largely on biological interactions, that attempt to explain within assemblage structure and succession were not broadly applicable.     

The chromosome partitioning protein, ParB, is required for cytokinesis in Caulobacter crescentus

In Caulobacter crescentus, the genes encoding the chromosome partitioning proteins, ParA and ParB, are essential. Depletion of ParB resulted in smooth filamentous cells in which DNA replication continued. Immunofluorescence microscopy revealed that the formation of FtsZ rings at the mid-cell, the earliest molecular event in the initiation of bacterial cell division, was blocked in cells lacking ParB. ParB binds sequences near the C. crescentus origin of replication. Cell cycle experiments show that the formation of bipolarly localized ParB foci, and presumably localization of the origin of replication to the cell poles, preceded the formation of FtsZ rings at the mid-cell by 20 min. These results suggest that one major function of ParA and ParB may be to regulate the initiation of cytokinesis in C. crescentus.     

Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display

Fine epitope mapping of therapeutically relevant monoclonal antibodies (mAbs) specific for the epidermal growth factor receptor (EGFR) was accomplished through random mutagenesis and yeast surface display. Using this method, we have identified key residues energetically important for the binding of EGFR to the mAbs 806, 225, and 13A9. A yeast-displayed library of single point mutants of an EGFR ectodomain fragment (residues 273-621) was constructed by random mutagenesis and was screened for reduced binding to EGFR mAbs. If an EGFR mutant showed loss of binding to a mAb, this suggested that the mutated residue was potentially a contact residue. The mAb 806 binding epitope was localized to one face of a loop comprised of EGFR residues Cys287-Cys302, which is constrained by a disulfide bond and two salt bridges. The mAb 806 epitope as identified here is not fully accessible in the autoinhibited EGFR monomer conformation, which is consistent with the hypothesis that mAb 806 binds to a transitional form of EGFR as it changes from an autoinhibited to extended monomer. The amino acids Lys465 and Ile467 were identified as energetic hot spot residues for mAb 225 binding to EGFR. These residues are adjacent to the EGFR ligand-binding site, which is consistent with the ability of mAb 225 to block binding of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) ligands. Ser468 and Glu472 were identified as energetically important for mAb 13A9 binding to EGFR, and the location of this epitope suggests that mAb 13A9 mediates observed TGF-alpha blocking effects through conformational perturbation of EGFR domain III. Combinatorial library screening of yeast-displayed mutagenic proteins is a novel method to identify discontinuous and heat-denaturable mAb binding epitopes with residue-level resolution.     

Discovery of a small-molecule inhibitor of the TRIP8b–HCN interaction with efficacy in neurons

Major depressive disorder is a critical public health problem with a lifetime prevalence of nearly 17% in the United States. One potential therapeutic target is the interaction between hyperpolarization-activated cyclic nucleotide–gated (HCN) channels and an auxiliary subunit of the channel named tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b). HCN channels regulate neuronal excitability in the mammalian hippocampus, and recent work has established that antagonizing HCN function rescues cognitive impairment caused by chronic stress. Here, we utilize a high-throughput virtual screen to find small molecules capable of disrupting the TRIP8b–HCN interaction. We found that the hit compound NUCC-0200590 disrupts the TRIP8b–HCN interaction in vitro and in vivo. These results provide a compelling strategy for developing new small molecules capable of disrupting the TRIP8b–HCN interaction.     

Are High-Impact Species Predictable? An Analysis of Naturalised Grasses in Northern Australia

Predicting which species are likely to cause serious impacts in the future is crucial for targeting management efforts, but the characteristics of such species remain largely unconfirmed. We use data and expert opinion on tropical and subtropical grasses naturalised in Australia since European settlement to identify naturalised and high-impact species and subsequently to test whether high-impact species are predictable. High-impact species for the three main affected sectors (environment, pastoral and agriculture) were determined by assessing evidence against pre-defined criteria. Twenty-one of the 155 naturalised species (14%) were classified as high-impact, including four that affected more than one sector. High-impact species were more likely to have faster spread rates (regions invaded per decade) and to be semi-aquatic. Spread rate was best explained by whether species had been actively spread (as pasture), and time since naturalisation, but may not be explanatory as it was tightly correlated with range size and incidence rate. Giving more weight to minimising the chance of overlooking high-impact species, a priority for biosecurity, meant a wider range of predictors was required to identify high-impact species, and the predictive power of the models was reduced. By-sector analysis of predictors of high impact species was limited by their relative rarity, but showed sector differences, including to the universal predictors (spread rate and habitat) and life history. Furthermore, species causing high impact to agriculture have changed in the past 10 years with changes in farming practice, highlighting the importance of context in determining impact. A rationale for invasion ecology is to improve the prediction and response to future threats. Although our study identifies some universal predictors, it suggests improved prediction will require a far greater emphasis on impact rather than invasiveness, and will need to account for the individual circumstances of affected sectors and the relative rarity of high-impact species.     

Survival and persistence of bioluminescent Xanthomonas campestris pv. campestris on host and non-host plants in the field environment

Dispersal and persistence of a pathogenic strain of Xanthomonas campestris pv. campestris , genetically engineered to bioluminesce, was followed in and on host and non-host plants in the field environment. Black rot susceptible cabbage plants were mist inoculated with the bioluminescent strain only, or were mist inoculated with X. campestris pv. vesicatoria or a weakly pathogenic strain of X. c. campestris 1 week before challenge inoculation with the bioluminescent strain. Growth of the bioluminescent strain was detected with a low-light, charge-coupled device camera or through bioluminescence measurements of broth-enrichment cultures of leaf disk samples. Bioluminescent X. c. campestris could often be observed as populations on symptomless leaves or in lesions, and persisted as a vascular endophyte for more than 6 months throughout the winter growing season. Dispersal to cruciferous and non-cruciferous weeds was frequently detected. Pre-inoculation with X. c. vesicatoria or the weakly pathogenic X. c. campestris did not significantly affect the movement and persistence of the bioluminescent strain nor reduce the incidence of black rot disease.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens . It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus . It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43°C-45°C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10–20 μg/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens .
The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfringens could be obtained by pre-incubation at 37°C of inoculated media for 2–4 h followed by overnight incubation at 43°C-45°C. Tryptose-sulphite-cyclo-serine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens . This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus . Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clos-tridia on BCP is presented.     

Activity and specificity of covalently immobolized wheat germ agglutinin toward cell surfaces

Wheat germ agglutinin protein, which is able to agglutinate tumor cells better than normal cells, was covalently bound to polyacrylamide gel beads. The specific binding activity of the protein was preserved on these beads and was expressed heterogeneously by the binding of mouse leukemia cells (L1210) to the protein coupled gels. The selective activity of the immobilized protein was maximal when the number of sites available to covalently couple the protein was lowest. The application of this observation to the general field of covalent immobilization of proteins and enzymes may be of considerable utility.     

Improved chemical analysis of cellulose ethers using dialkylamine derivatization and mass spectrometry

Oligosaccharides of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and methyl cellulose were investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The cellulose ether oligosaccharides were produced either by enzymatic depolymerization utilizing the purified family 5 endoglucanase from Bacillus agaradhaerens or by partial acidic depolymerization. To lower the limit of detection in MALDI-MS three dilakylamines, dimethyl-, diethyl-, and dipropylamine were studied as reagents for reductive amination of the oligosaccharides. All three amines contributed to a significant increase in sensitivity in MALDI-MS, especially for oligosaccharides with a degree of polymerization (DP) < 3. These reagents were also attractive due to their high volatility, which facilitated the purification of the reaction mixtures. It was established that low-mass discrimination in MALDI-MS in the DP range 1-7 was substantially reduced with dialkylamine derivatization. Hence, dialkylamine derivatization of cellulose ether oligosaccharides obtained by endoglucanase depolymerization increased the number of detected analyte components. Dimethylamine was concluded to be the preferred reagent of those evaluated.