Proteomic analysis of nicotine-associated protein expression in the striatum of repeated nicotine-treated rats

Through the proteomic analysis using 2-dimensional electrophoresis, the nicotine addiction-associated proteins were extensively screened in the striatum of rat brains. The nicotine addiction was developed by repeated nicotine injection (0.4mg/kg s.c.), twice daily for 7 days, followed by one challenge injection after a 3 day withdrawal period, and then confirmed by observing a 2.3-fold increase in locomoter activity. The 3 up- and 4 down-regulated proteins were selected and identified to be zinc-finger binding protein-89 (ZBP-89), 2'3'-cyclic nucleotide 3'-phosphodiesterase 1, deoxyribonuclease 1-like 3 (DNase1l3), tandem pore domain halothane inhibited K(+) channel (THIK-2), brain-specific hyaluronan-binding protein (BRAL-1), death effector domain-containing DNA binding protein (DEDD), and brain-derived neurotrophic factor (BDNF) by mass spectrophotometric fingerprinting. Among them, the expression patterns of ZEB-89, DNase1l3, THIK-2, DEDD, and BDNF mRNAs were found to be coincident with those of cognate proteins, by using RT-PCR analysis. These proteins could be suggested as drug targets to develop a new therapy for nicotine-associated diseases, as well as the clues to understand the mechanism of nicotine.     

Genomic segments cloning and analysis of Cotesia plutellae polydnavirus using plasmid capture system

Cotesia plutellae polydnaviruses (CpBV) has a segmented genome consisting of multiple circular double stranded DNAs. Recently, we have developed an easy, simple, and convenient system based on Tn7 transposition in order to clone genomic segments of CpBV in Escherichia coli cell and designated plasmid capture system (PCS). The PCS donor-S transferred a pUC19 origin of replication and an ampicillin resistance marker into CpBV genomic DNA by in vitro transposition. Through PCS system, we were able to clone 53 genomic clones ranging from 0.1 to 25.5 kb and further they were classified into 29 segments by their sizes and restriction endonuclease patterns. Among them, a complete nucleotide sequence of CpBV-S28 segment was determined and 10 putative genes were predicted from this segment. Interestingly, 9 of 10 putative ORFs had high level of similarities with catalytic domain of protein tyrosine phosphatase. Also, ORF2807 showed similarity with EP1-like proteins of C. congregata polydnavirus.     

Estrogen receptor beta in health and disease

Estrogens, acting through its two receptors, ESR1 (hereafter designated ER alpha) and ESR2 (hereafter designated ER beta), have diverse physiological effects in the reproductive system, bone, cardiovascular system, hematopoiesis, and central and peripheral nervous systems. Mice with inactivated ER alpha, ER beta, or both show a number of interesting phenotypes, including incompletely differentiated epithelium in tissues under steroidal control (prostate, ovary, mammary, and salivary glands) and defective ovulation reminiscent of polycystic ovarian syndrome in humans (in ER beta-/- mice), and obesity, insulin resistance, and complete infertility (both in male and female ER alpha-/- mice). Estrogen agonists and antagonists are frequently prescribed drugs with indications that include postmenopausal syndrome (agonists) and breast cancer (antagonists). Because the two estrogen receptors (ERs) have different physiological functions and have ligand binding pockets that differ enough to be selective in their ligand binding, opportunities now exist for development of novel ER subtype-specific selective-ER modulators.     

Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling

The auditory sensory epithelium (organ of Corti), where sound waves are converted to electrical signals, comprises a highly ordered array of sensory receptor (hair) cells and nonsensory supporting cells. Here, we report that Sprouty2, which encodes a negative regulator of signaling via receptor tyrosine kinases, is required for normal hearing in mice, and that lack of SPRY2 results in dramatic perturbations in organ of Corti cytoarchitecture: instead of two pillar cells, there are three, resulting in the formation of an ectopic tunnel of Corti. We demonstrate that these effects are due to a postnatal cell fate transformation of a Deiters' cell into a pillar cell. Both this cell fate change and hearing loss can be partially rescued by reducing Fgf8 gene dosage in Spry2 null mutant mice. Our results provide evidence that antagonism of FGF signaling by SPRY2 is essential for establishing the cytoarchitecture of the organ of Corti and for hearing.     

Evolution of aldolase antibodies in vitro: correlation of catalytic activity and reaction-based selection

Aldolase antibodies that operate via an enamine mechanism were developed by in vitro selection. Antibody Fab phage display libraries were created where the catalytic active site residues of aldolase antibodies 38C2 and 33F12 were combined with a naive human antibody V gene repertoire. Selection from these libraries with 1,3-diketones covalently trapped the amino groups of reactive lysine residues by formation of stable enaminones. The selected aldolase antibodies retained the essential catalytic lysine residue and its function in altered and humanized primary antibody structures. The substrate specificity of the aldolase antibodies was directly related to the structure of the diketone used for selection. The k(cat) values of the antibody-catalyzed retro-aldol reactions were correlated with the K(d) values, i.e. the reactivities of the selected aldolase antibodies for the corresponding diketones. Antibodies that bound to the diketone with a lower K(d) value displayed a higher k(cat) value in the retro-aldol reaction, and a linear relationship was observed in the plots of logk(cat) versus logK(d). These results indicate that selections with diketones directed the evolution of aldolase antibodies in vitro that operate via an enamine mechanism. This strategy provides a route to tailor-made aldol catalysts with different substrate specificities.     

Magnesium influences the discrimination and release of ADP by human RAD51

hRAD51 lacks cooperative DNA-dependent ATPase activity and appears to function with 5-10-fold less Mg2+ compared to RecA. We have further explored the effect of Mg2+ on adenosine nucleotide binding, ATPase, and DNA strand exchange activities. hRAD51 was saturated with the poorly hydrolyzable analog of ATP, ATPgammaS, at approximately 0.08 mM Mg2+. In contrast, > 0.5 mM Mg2+ was required to saturate hRAD51 with ADP. We found ADP to be a significantly less effective competitive inhibitor of the hRAD51 ATPase at low Mg2+ concentrations (0.08 mM). Mg2+ did not appear to affect the ability of ATPgammaS to competitively inhibit the hRAD51 ATPase. Low Mg2+ (0.08-0.12 mM) enhanced the steady-state ATPase of hRAD51 while higher Mg2+ concentration (> 0.3 mM) was inhibitory. At low Mg2+, hRAD51 appeared capable of nearly complete hydrolysis of available ATP, suggesting a lack of ADP product inhibition. There was a strong correlation between the amount of Mg2+ required for stable ADP binding and the inhibition of hRad51 strand exchange activity. Simultaneous inclusion of exogenous ATP and chelation of Mg2+ with EDTA significantly enhanced ADP-->ATP exchange by hRAD51. These studies are consistent with the hypothesis that Mg2+ influences the discrimination and release of ADP, which may sequentially impose an important regulatory step in the hRAD51 ATPase cycle.     

Optimization of free ammonia concentration for nitrite accumulation in shortcut biological nitrogen removal process

A shortcut biological nitrogen removal (SBNR) utilizes the concept of a direct conversion of ammonium to nitrite and then to nitrogen gas. A successful SBNR requires accumulation of nitrite in the system and inhibition of the activity of nitrite oxidizers. A high concentration of free ammonia (FA) inhibits nitrite oxidizers, but unfortunately decreases the ammonium removal rate as well. Therefore, the optimal range of FA concentration is necessary not only to stabilize nitrite accumulation but also to achieve maximum ammonium removal. In order to derive such optimal FA concentrations, the specific substrate utilization rates of ammonium and nitrite oxidizers were measured. The optimal FA concentration range appeared to be 5–10 mg/L for the adapted sludge. The simulated results from the modified inhibition model expressed by FA and ammonium/nitrite concentrations were shown very similar to the experimental results.     

Systematically assessing the influence of 3-dimensional structural context on the molecular evolution of mammalian proteomes

The 3-dimensional (3D) structural context of amino acid residues in a protein could significantly impact the level of selective constraint on the residues. Here, by analyzing 767 mammalian proteins, we systematically investigate how various 3D structural contexts influence selective constraint. The structural contexts we examined include solvent accessibility, secondary structure, and intramolecular residue-residue interactions. Through this analysis, we offer quantitative information on how 3D structural contexts affect the level of selective constraint.