NMR based metabonomics study of NPY Y5 receptor activation in BT-549, a human breast carcinoma cell line

Overexpression of neuropeptide Y (NPY) and its receptors has been found in various cancers. In our previous study, we demonstrated expression of NPY Y5 receptor (Y5R) in various breast cancer cell lines along with Y1 receptor. In Y5R expressing BT-549 cells, NPY induced cell proliferation that was blocked by Y5R-selective antagonist CGP1683A (CGP). Here, NMR-based metabonomics was used to monitor the metabolic profile of BT-549 cells in the presence of NPY and CGP to assess the effect of Y5R activation and inhibition during NPY-induced cell proliferation. To study changes in intra and extra cellular metabolites in response to various treatments, 1D ¹H-NMR spectra of both hydrophilic cell extracts and growth medium were recorded from BT-549 with three treatments: (1) NPY, (2) CGP, and (3) CGP followed by NPY (CGP/NPY). Principal component analysis and statistical significance analysis indicated changes in intracellular concentrations of seven metabolites in hydrophilic cell extracts with NPY treatment: decreases in lactate, succinate, myo-inositol, and creatine, and increases in acetate, glutamate, and aspartate. A significant increase in intracellular lactate level and attenuation of other metabolites to baseline was detected in CGP/NPY group. Also, significant decreases in lactate and increases in pyruvate were observed in growth medium from NPY treated cells. Based on the metabonomics analysis, Y5R activation induces cell proliferation by increasing the rate of glycolysis, glutaminolysis, and TCA cycle. Inhibition of Y5R by CGP counteracts NPY-induced changes in cellular metabolites. These changes may play a role in cell proliferation and migration by NPY through Y5R activation.     

A procedure to recruit members to enlarge protein family databases--the building of UECOG (UniRef-Enriched COG Database) as a model

A procedure to recruit members to enlarge protein family databases is described here. The procedure makes use of UniRef50 clusters produced by UniProt. Current family entries are used to recruit additional members based on the UniRef50 clusters to which they belong. Only those additional UniRef50 members that are not fragments and whose length is within a restricted range relative to the original entry are recruited. The enriched dataset is then limited to contain only genomes from selected clades. We used the COG database - used for genome annotation and for studies of phylogenetics and gene evolution - as a model. To validate the method, a UniRef-Enriched COG0151 (UECOG) was tested with distinct procedures to compare recruited members with the recruiters: PSI-BLAST, secondary structure overlap (SOV), Seed Linkage, COGnitor, shared domain content, and neighbor-joining single-linkage, and observed that the former four agree in their validations. Presently, the UniRef50-based recruitment procedure enriches the COG database for Archaea, Bacteria and its subgroups Actinobacteria, Firmicutes, Proteobacteria, and other bacteria by 2.2-, 8.0-, 7.0-, 8.8-, 8.7-, and 4.2-fold, respectively, in terms of sequences, and also considerably increased the number of species.     

Role of the carboxy-termini of tubulin on its chaperone-like activity.

Mutational analysis and the enzymatic digestion of many chaperones indicate the importance of both hydrophobic and hydrophilic residues for their unique property. Thus, the chaperone activity of alpha-crystallin is lost due to the substitution of hydrophobic residues or upon enzymatic digestion of the negatively charged residues. Tubulin, an eukaryotic cytoskeletal protein, exhibits chaperone-like activity as demonstrated by prevention of DTT-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase, betagamma-crystallin, and other proteins. We have shown that the tubulin lost its chaperone-like activity upon digestion of its negatively charged C-termini. In this article, the role of the C-terminus of individual subunits has been investigated. We observe that the digestion of C-terminus of beta-subunit with subtilisin causes loss of chaperone-like activity of tubulin. The contribution of C-terminus of alpha-subunit is difficult to establish directly as subtilisin cleaves C-terminus of beta-subunit first. This has been ascertained indirectly using a 14-residue peptide P2 having the sequence corresponding to a conserved region of MHC class I molecules and that binds tightly to the C-terminus of alpha-subunit. We have shown that the binding of P2 peptide to alphabeta-tubulin causes complete loss of its chaperone-like activity. NMR and gel-electrophoresis studies indicate that the P2 peptide has a significant higher binding affinity for the C-terminus of alpha-subunit compared to that of beta-subunit. Thus, we conclude that both the C-termini are necessary for the chaperone-like activity of tubulin. Implications for the chaperone functions in vivo have been discussed.     

Rapid and high-throughput analysis of N-glycans from ovarian cancer serum using a 96-well plate platform

We present a rapid and high-throughput human serum N-glycan preparation technology using 96-well plate-based procedures. The released N-glycans from polyvinylidene fluoride (PVDF) membrane filter plate are subsequently loaded to porous graphitic carbon (PGC) containing a 96-well plate to remove salts and other contaminants without sacrificing accuracy or reproducibility. This robust glycan preparation technology is applied to ovarian cancer diagnosis using 5 μl of patient serum.     

Helicobacter pylori treatment: antibiotics or probiotics

Applied Microbiology and Biotechnology - Treatment of Helicobacter pylori infection is important for the management of gastrointestinal disorders such as peptic ulcer and gastric cancer. Due to the...     

Prp19/Pso4 Is an Autoinhibited Ubiquitin Ligase Activated by Stepwise Assembly of Three Splicing Factors

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Dynamics of spontaneous activity in random networks with multiple neuron subtypes and synaptic noise

During slow-wave sleep, brain electrical activity is dominated by the slow (< 1 Hz) electroencephalogram (EEG) oscillations characterized by the periodic transitions between active (or Up) and silent (or Down) states in the membrane voltage of the cortical and thalamic neurons. Sleep slow oscillation is believed to play critical role in consolidation of recent memories. Past computational studies, based on the Hodgkin-Huxley type neuronal models, revealed possible intracellular and network mechanisms of the neuronal activity during sleep, however, they failed to explore the large-scale cortical network dynamics depending on collective behavior in the large populations of neurons. In this new study, we developed a novel class of reduced discrete time spiking neuron models for large-scale network simulations of wake and sleep dynamics. In addition to the spiking mechanism, the new model implemented nonlinearities capturing effects of the leak current, the Ca²⁺ dependent K⁺ current and the persistent Na⁺ current that were found to be critical for transitions between Up and Down states of the slow oscillation. We applied the new model to study large-scale two-dimensional cortical network activity during slow-wave sleep. Our study explained traveling wave dynamics and characteristic synchronization properties of transitions between Up and Down states of the slow oscillation as observed in vivo in recordings from cats. We further predict a critical role of synaptic noise and slow adaptive currents for spike sequence replay as found during sleep related memory consolidation.     

Evaluation of cDNA Libraries from Different Developmental Stages of Schistosoma mansoni for Production of Expressed Sequence Tags (ESTs)

A comparative study of the gene expression profile in differentdevelopmental stages of Schistosoma mansoni has been initiatedbased on the expressed sequence tag(EST) approach. A total of1401 ESTs were generated from seven different cDNA librariesconstructed from four distinct stages of the parasite life cycle.The libraries were first evaluated for their quality for a large-scalecDNA sequencing program. Most of them were shown to have lessthan 20% useless clones and more than 50% new genes. The redundancyof each library was also analyzed, showing that one adult wormcDNA library was composed of a small number of highly frequentgenes. When comparing ESTs from distinct libraries, we coulddetect that most genes were present only in a single library,but others were expressed in more than one developmental stageand may represent housekeeping genes in the parasite. When consideringonly once the genes present in more than one library, a totalof 466 unique genes were obtained, corresponding to 427 newS. mansoni genes. From the total of unique genes, 20.2% wereidentified based on homology with genes from other organisms,8.3% matched S. mansoni characterized genes and 71.5% representunknown genes.