Identification of novel kinase inhibitors by targeting a kinase-related apoptotic protein-protein interaction network in HeLa cells.

OBJECTIVES: Protein kinases orchestrate activation of signalling cascades in response to extra- and intracellular stimuli for regulation of cell proliferation. They are directly involved in a variety of diseases, particularly cancers. Systems biology approaches have become increasingly important in understanding regulatory frameworks in cancer, and thus may facilitate future anti-cancer discoveries. Moreover, it has been suggested and confirmed that high-throughput virtual screening provides a novel, effective way to reveal small molecule protein kinase inhibitors. Accordingly, we aimed to identify kinase targets and novel kinase inhibitors. MATERIALS AND METHODS: A series of bioinformatics methods, such as network construction, molecular docking and microarray analyses were performed. RESULTS: In this study, we computationally constructed the appropriate global human protein-protein interaction network with data from online databases, and then modified it into a kinase-related apoptotic protein-protein interaction network. Subsequently, we identified several kinases as potential drug targets according to their differential expression observed by microarray analyses. Then, we predicted relevant microRNAs, which could target the above-mentioned kinases. Ultimately, we virtually screened a number of small molecule natural products from Traditional Chinese Medicine (TCM)@Taiwan database and identified a number of compounds that are able to target polo-like kinase 1, cyclin-dependent kinase 1 and cyclin-dependent kinase 2 in HeLa cervical carcinoma cells. CONCLUSIONS: Taken together, all these findings might hopefully facilitate discovery of new kinase inhibitors that could be promising candidates for anti-cancer drug development.

Identifying promising compounds in drug discovery: genetic algorithms and some new statistical techniques.

Throughout the drug discovery process, discovery teams are compelled to use statistics for making decisions using data from a variety of inputs. For instance, teams are asked to prioritize compounds for subsequent stages of the drug discovery process, given results from multiple screens. To assist in the prioritization process, we propose a desirability function to account for a priori scientific knowledge; compounds can then be prioritized based on their desirability scores. In addition to identifying existing desirable compounds, teams often use prior knowledge to suggest new, potentially promising compounds to be created in the laboratory. Because the chemistry space to search can be dauntingly large, we propose the sequential elimination of level combinations (SELC) method for identifying new optimal compounds. We illustrate this method on a combinatorial chemistry example.

Spontaneous acetylcholine secretion from developing growth cones of Drosophila central neurons in culture: effects of cAMP-pathway mutations.

We describe a novel bioassay system that uses Xenopus embryonic myocytes (myoballs) to detect the release of acetylcholine from Drosophila CNS neurons. When a voltage-clamped Xenopus myoball was manipulated into contact with cultured Drosophila "giant" neurons, spontaneous synaptic current-like events were registered. These events were observed within seconds after contact and were blocked by curare and alpha-bungarotoxin, but not by TTX and Cd(2+), suggesting that they are caused by the spontaneous quantal release of acetylcholine (ACh). The secretion occurred not only at the growth cone, but also along the neurite and at the soma, with significantly different release parameters among various regions. The amplitude of these currents displayed a skewed distribution. These features are distinct from synaptic transmission at more mature synapses or autapses formed in this culture system and are reminiscent of the transmitter release process during early development in other preparations. The usefulness of this coculture system in studying presynaptic secretion mechanisms is illustrated by a series of studies on the cAMP pathway mutations, dunce (dnc) and PKA-RI, which disrupt a cAMP-specific phosphodiesterase and the regulatory subunit of cAMP-dependent protein kinase A, respectively. We found that these mutations affected the ACh current kinetics, but not the quantal ACh packet, and that the release frequency was greatly enhanced by repetitive neuronal activity in dnc, but not wild-type, growth cones. These results suggest that the cAMP pathway plays an important role in the activity-dependent regulation of transmitter release not only in mature synapses as previously shown, but also in developing nerve terminals before synaptogenesis.

Protective effects of pseudoginsenoside-F11 on scopolamine-induced memory impairment in mice and rats.

This study assessed the effects of pseudoginsenoside-F11, a component of Panax quinquefolium L., on scopolamine-impaired memory performance in mice and rats. In the one-trial step-down and step-through passive avoidance tests, although pseudo-ginsenoside-F11 used alone did not affect passive avoidance behaviour in naive mice, the latency of avoidance shortened by intraperitoneal scopolamine (2 mg kg(-1)) was prolonged after intragastric administration of pseudoginsenoside-F11 (2 or 4 mg kg(-1), for five days) in both test systems in mice. In the water-maze test, in mice, the time taken to locate the platform after administration of pseudoginsenoside-F11 was shorter than that after administration of scopolamine (1 mg kg(-1), i.p.). In the two-way active avoidance response test, the latency of avoidance was significantly shorter for the pseudoginsenoside-F11-(1.2 or 2.4 mg kg(-1), i.g. for five days) and scopolamine-treated group than for the group of rats given scopolamine only (2 mg kg(-1), i.p.). The percentage avoidance was also reduced after intraperitoneal injection of scopolamine, but was reversed by administration of pseudo-ginsenoside-F11. These results suggest that pseudoginsenoside-F11 antagonized the memory dysfunction induced by scopolamine. However, the mechanism of the memory facilitative action of pseudoginsenoside-F11 merits further elucidation.

Functional expression of Shaker K+ channels in cultured Drosophila "giant" neurons derived from Sh cDNA transformants: distinct properties, distribution, and turnover.

Expression of transgenic Shaker (Sh) channels has not previously been examined in Drosophila neurons. We studied K+ current by whole-cell recording in cultured "giant" neurons derived from germline transformants. Independent lines were generated by using a P-element vector, in which transcription of the 29-4 cDNA, one of the Sh splicing variants (Iverson and Rudy, 1990), was under the control of a heat shock (HS)-inducible promoter. Transformants in wild-type and two different Sh mutant backgrounds all exhibited an HS-inducible, A-type K+ current that was characterized by a much slower recovery from inactivation and a higher sensitivity to 4-aminopyridine than native K+ currents of Sh 29-4 currents expressed in Xenopus oocytes. Despite similarities in the kinetic and pharmacological properties of the HS-induced current in all backgrounds examined, host-dependent differences in the peak current amplitude have been consistently observed between multiple lines of 29-4 ShM and 29-4 Sh120 that might reflect differential channel subunit assembly in different hosts. Isolation of the novel 29-4 currents allowed determination of the channel turnover rate in cultured neurons. These currents persisted for up to 3 d or more, comparable with the durations previously reported for Na+ and Ca2+ channels. Surprisingly, the percentage of cells expressing inactivating K+ currents remained approximately the same with or without HS induction, suggesting that some mechanisms exist to restrict functional expression of inactivating K+ channels, including transgenic Sh channels and those not encoded by the Sh locus, to certain types of neurons.

Removal of nitrogen and phosphorus from swine wastewater by intermittent aeration processes.

Nitrogen and phosphorus removal in a methane fermentation plus activated sludge method type pig farm (M-III) with an intermittent aeration process (IAP) was evaluated in comparison with a continuous aeration process (CAP) based on the full-scale and bench-scale experiments. Operation conditions for the treatment system were the same except for the aeration program (in the CAP), a consecutive 24-hr aeration was used, whereas in the IAP, the aeration and non-aeration periods were alternated at intervals of 3:1 hr. BOD and TOC removal efficiencies with the intermittent aeration were as high as those with the continuous operation (92-98%). In the removal of nitrogen and phosphorus, large differences between IAP and CAP were observed. At an influent T-N/TOC ratio of 0.1, removal efficiencies for T-N in the bench-scale IAP was 70%, and for T-P was 22%, respectively. At an even higher influent T-N/TOC ratios of 0.3-1.0, the removal efficiencies for T-N were decreased to about 59-61%, whereas that for T-P were -0.3-13%. In a full-scale plant, removal efficiencies for T-N with IAP and CAP were 42% and -0.09%, respectively. The results of this study show the successful performance of a simple IAP for piggery wastewater: simultaneous, one-sludge denitrification with nitrification in single-activated sludge reactor in a pig farm.