Using capillary electrophoresis with laser-induced fluorescence to study the interaction of green fluorescent protein-labeled calmodulin with Ca2+- and calmodulin-binding protein

A separation using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was applied to the study of green fluorescent protein tagged calmoldulin (GFP-CaM) that was expressed from Escherichia coli and purified with Ni(2+)-nitrilotriacetate (Ni-NTA) resin column. It was found that GFP-CaM not only has good fluorescence properties under various conditions similar to GFP, but also retains its calcium-binding ability as the native CaM. GFP-CaM was separated and detected by CE-LIF within 10 min with a limit-of-detection (LOD) of 2 x 10(-10) M for an injection volume of 3 nl, higher than that of common chemical fluorescent-tagged protein method. The results indicated that, as a fluorescence probe, GFP could overcome the drawback of inefficient derivatization of chemical fluorescence probes. The interaction between the GFP-CaM and Ca(2+) was studied in detail using affinity capillary electrophoresis with laser-induced fluorescence and the dissociation constant (K(d)) between GFP-CaM and Ca(2+) was determined to be 1.2 x 10(-5), which is in good agreement with the literature values of untagged CaM (10(-6) to 10(-5)M) obtained by conventional method. As a preliminary application, the interaction between GFP-CaM and OsCBK was also investigated. The method makes it possible to screen the trace amounts of target proteins in crude extracts interacting with CaM under physiological conditions.     

Molecular classification of liver cirrhosis in a rat model by proteomics and bioinformatics

Liver cirrhosis is a worldwide health problem. Reliable, noninvasive methods for early detection of liver cirrhosis are not available. Using a three-step approach, we classified sera from rats with liver cirrhosis following different treatment insults. The approach consisted of: (i) protein profiling using surface-enhanced laser desorption/ionization (SELDI) technology; (ii) selection of a statistically significant serum biomarker set using machine learning algorithms; and (iii) identification of selected serum biomarkers by peptide sequencing. We generated serum protein profiles from three groups of rats: (i) normal (n=8), (ii) thioacetamide-induced liver cirrhosis (n=22), and (iii) bile duct ligation-induced liver fibrosis (n=5) using a weak cation exchanger surface. Profiling data were further analyzed by a recursive support vector machine algorithm to select a panel of statistically significant biomarkers for class prediction. Sensitivity and specificity of classification using the selected protein marker set were higher than 92%. A consistently down-regulated 3495 Da protein in cirrhosis samples was one of the selected significant biomarkers. This 3495 Da protein was purified on-chip and trypsin digested. Further structural characterization of this biomarkers candidate was done by using cross-platform matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting (PMF) and matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS). Combined data from PMF and MS/MS spectra of two tryptic peptides suggested that this 3495 Da protein shared homology to a histidine-rich glycoprotein. These results demonstrated a novel approach to discovery of new biomarkers for early detection of liver cirrhosis and classification of liver diseases.     

Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions

We have determined the structure of a mutant (Q237W) of HhaI DNA methyltransferase, complexed with the methyl-donor product AdoHcy. The Q237W mutant proteins were crystallized in the monoclinic space group C2 with two molecules in the crystallographic asymmetric unit. Protein-protein interface calculations in the crystal lattices suggest that the dimer interface has the specific characteristics for homodimer protein-protein interactions, while the two active sites are spatially independent on the outer surface of the dimer. The solution behavior suggests the formation of HhaI dimers as well. The same HhaI dimer interface is also observed in the previously characterized binary (M.HhaI-AdoMet) and ternary (M.HhaI-DNA-AdoHcy) complex structures, crystallized in different space groups. The dimer is characterized either by a non-crystallographic two-fold symmetry or a crystallographic symmetry. The dimer interface involves three segments: the amino-terminal residues 2-8, the carboxy-terminal residues 313-327, and the linker (amino acids 179-184) between the two functional domains--the catalytic methylation domain and the DNA target recognition domain. Both the amino- and carboxy-terminal segments are part of the methylation domain. We also examined protein-protein interactions of other structurally characterized DNA MTases, which are often found as a 2-fold related 'dimer' with the largest dimer interface area for the group-beta MTases. A possible evolutionary link between the Type I and Type II restriction-modification systems is discussed.     

Optical spectroscopy to investigate the structure of regenerated Bombyx mori silk fibroin in solution

Fluorescence and circular dichroism spectroscopy were used to monitor the conformational transition of regenerated Bombyx mori silk fibroin (RSF) in aqueous solutions under different conditions. According to the analysis of fluorescence spectra using anilinonaphthalene-8-sulfonic acid magnesium salt (ANS) as an external probe, the destruction of the hydrophobic core prior to the secondary structure change suggests that this collapse may initiate the conformational transition from random coil to beta-sheet for RSF. The temperature dependence of the structural changes of RSF, detected by both fluorescence spectroscopy and circular dichroism, shows a reversible process upon heating and recooling, with the midpoint around 45 degrees C. The results also indicate that most of the tryptophan (Trp) residues contained in silk fibroin are concentrated on the surface of the unfolded protein. However, they will change their location in the highly ordered structure (e.g., becoming more homogeneous) with the conformational transition of silk fibroin. Moreover, our studies also suggest that the presence of water plays a crucial role during the structure changes of fibroin.     

Deletion of the neuron-specific protein delta-catenin leads to severe cognitive and synaptic dysfunction

Delta-catenin (delta-catenin) is a neuron-specific catenin, which has been implicated in adhesion and dendritic branching. Moreover, deletions of delta-catenin correlate with the severity of mental retardation in Cri-du-Chat syndrome (CDCS), which may account for 1% of all mentally retarded individuals. Interestingly, delta-catenin was first identified through its interaction with Presenilin-1 (PS1), the molecule most frequently mutated in familial Alzheimer's Disease (FAD). We investigated whether deletion of delta-catenin would be sufficient to cause cognitive dysfunction by generating mice with a targeted mutation of the delta-catenin gene (delta-cat(-/-)). We observed that delta-cat(-/-) animals are viable and have severe impairments in cognitive function. Furthermore, mutant mice display a range of abnormalities in hippocampal short-term and long-term synaptic plasticity. Also, N-cadherin and PSD-95, two proteins that interact with delta-catenin, are significantly reduced in mutant mice. These deficits are severe but specific because delta-cat(-/-) mice display a variety of normal behaviors, exhibit normal baseline synaptic transmission, and have normal levels of the synaptic adherens proteins E-cadherin and beta-catenin. These data reveal a critical role for delta-catenin in brain function and may have important implications for understanding mental retardation syndromes such as Cri-du-Chat and neurodegenerative disorders, such as Alzheimer's disease, that are characterized by cognitive decline.     

Design of bivalent ligands using hydrogen bond linkers: synthesis and evaluation of inhibitors for human beta-tryptase

We exploit the concept of using hydrogen bonds to link multiple ligands for maintaining simultaneous interactions with polyvalent binding sites. This approach is demonstrated by the syntheses and evaluation of pseudo-bivalent ligands as potent inhibitors of human β-tryptase.     

Design, synthesis, and biological evaluation of novel 7-azaindolyl-heteroaryl-maleimides as potent and selective glycogen synthase kinase-3beta (GSK-3beta) inhibitors

Two approaches were developed to synthesize the novel 7-azaindolyl-heteroarylmaleimides. The first approach was based upon the palladium-catalyzed Suzuki cross-coupling or Stille cross-coupling of 2-chloro-maleimide 5 with various arylboronic acids or arylstannanes. The second approach was based upon the condensation of ethyl 7-azaindolyl-3-glyoxylate 12 with various acetamides. The hydroxypropyl-substituted 7-azaindolylmaleimide template was first used to screen different heteroaryls attached to the maleimide. Replacement of hydroxypropyl with different chain lengths and different functional groups were studied next. Many compounds synthesized were demonstrated to have high potency at GSK-3beta, good GS activity in HEK293 cells and good to excellent metabolic stability in human liver microsomes. Three representative compounds (21, 33, and 34) were demonstrated to have good selectivity against a panel of 80 kinase assays. Among them, compound 33 exhibited very weak inhibitions at the other 79 kinase assays, and behaved as a highly selective GSK-3beta inhibitor.     

重组腺相关病毒转染神经干细胞球的实验研究

目的:探讨重组腺相关病毒2型(rAAV2)对神经干细胞球的转染能力.方法:①将FITC标记的rAAV2(FITC-rAAV2)分成两组,A组直接与神经干细胞球混合,B组与肝素混匀后再与神经干细胞球混合,孵育30 min后在荧光显微镜下观察;②含有GFP报告基因的rAAV2(rAAV2-GFP)与神经干细胞球孵育30 min后,分成两组:A组继续在培养箱内培养,B组分散成单细胞后移植到大鼠脑内,一个月后分别在荧光显微镜下观察神经干细胞球和大鼠脑组织切片中报告基因的表达情况;③将含有低氧启动子(低氧应答元件,HRE)、VEGF和GFP的rAAV2(rAAV2-HRE-VEGF-GFP)转染神经干细胞球后分为两组:A组在低氧条件下培养,B组在常规条件下培养,72 h后观察报告基因的表达情况.结果:①FITC-rAAV2转染神经干细胞球的结果:A组有明亮的绿色荧光,B组基本无绿色荧光;②rAAV2-GFP转染神经干细胞球后一个月,A、B两组均可以看到绿色荧光;③rAAV2-HRE-VEGF-GFP转染神经干细胞球后72 h,A组可见绿色荧光,B组无绿色荧光.结论:rAAV2可以与神经干细胞球特异性结合,rAAV2携带的外源基因在体内和体外均可以有效表达,rAAV2携带外源基因的表达可以人为调控.