Direct electron transfer and enzymatic activity of hemoglobin in a hexagonal mesoporous silica matrix

The direct electrochemistry of hemoglobin (Hb) immobilized on a hexagonal mesoporous silica (HMS)-modified glassy carbon electrode was described. The interaction between Hb and the HMS was investigated using UV-Vis spectroscopy, FT-IR, and electrochemical methods. The direct electron transfer of the immobilized Hb exhibited two couples of redox peaks with the formal potentials of -0.037 and -0.232 V in 0.1 M (pH 7.0) PBS, respectively, which corresponded to its two immobilized states. The electrode reactions showed a surface-controlled process with a single proton transfer at the scan rate range from 20 to 200 mV/s. The immobilized Hb retained its biological activity well and displayed an excellent response to the reduction of both hydrogen peroxide (H2O2) and nitrate (NO2-). Its apparent Michaelis-Menten constants for H2O2 and NO2- were 12.3 and 49.3 microM, respectively, showing a good affinity. Based on the immobilization of Hb on the HMS and its direct electrochemistry, two novel biosensors for H2O2 and NO2- were presented. Under optimal conditions, the sensors could be used for the determination of H2O2 ranging from 0.4 to 6.0 microM and NO2- ranging from 0.2 to 3.8 microM. The detection limits were 1.86 x 10(-9) M and 6.11 x 10(-7) M at 3sigma, respectively. HMS provided a good matrix for protein immobilization and biosensor preparation.     

Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis

The characterization of protein modifications is essential for the study of protein function using functional genomic and proteomic approaches. However, current techniques are not efficient in determining protein modifications. We report an approach for sequencing proteins and determining modifications with high speed, sensitivity and specificity. We discovered that a protein could be readily acid-hydrolyzed within 1 min by exposure to microwave irradiation to form, predominantly, two series of polypeptide ladders containing either the N- or C-terminal amino acid of the protein, respectively. Mass spectrometric analysis of the hydrolysate produced a simple mass spectrum consisting of peaks exclusively from these polypeptide ladders, allowing direct reading of amino acid sequence and modifications of the protein. As examples, we applied this technique to determine protein phosphorylation sites as well as the sequences and several previously unknown modifications of 28 small proteins isolated from Escherichia coli K12 cells. This technique can potentially be automated for large-scale protein annotation.     

Signalling drought in guard cells

A number of environmental conditions including drought, low humidity, cold and salinity subject plants to osmotic stress. A rapid plant response to such stress conditions is stomatal closure to reduce water loss from plants. From an external stress signal to stomatal closure, many molecular components constitute a signal transduction network that couples the stimulus to the response. Numerous studies have been directed to resolving the framework and molecular details of stress signalling pathways in plants. In guard cells, studies focus on the regulation of ion channels by abscisic acid (ABA), a chemical messenger for osmotic stress. Calcium, protein kinases and phosphatases, and membrane trafficking components have been shown to play a role in ABA signalling process in guard cells. Studies also implicate ABA-independent regulation of ion channels by osmotic stress. In particular, a direct osmosensing pathway for ion channel regulation in guard cells has been identified. These pathways form a complex signalling web that monitors water status in the environment and initiates responses in stomatal movements.     

A novel GTP-binding protein hGBP3 interacts with NIK/HGK

A novel human guanylate-binding protein (GBP) hGBP3 was identified and characterized. Similar as the two human guanylate-binding proteins hGBP1 and hGBP2, hGBP3 has the first two motifs of the three classical guanylate-binding motifs, GXXXXGKS (T) and DXXG, but lacks the N (T) KXD motif. Escherichia coli-expressed hGBP3 protein specifically binds to guanosine triphosphate (GTP). Using a yeast two-hybrid system, it was revealed that the N-terminal region of hGBP3 binds to the C-terminal regulatory domain of NIK/HGK, a member of the group I GCK (germinal center kinase) family. This interaction was confirmed by in vitro glutathione-S-transferase (GST) pull-down and co-immunoprecipitation assays.     

CP27 affects viability,proliferation, attachment and gene expression in embryonic fibroblasts

CP27 is a gene that has been cloned from an E11 early embryonic library and has been suggested to mediate early organogenesis (Diekwisch et al., 1999, Gene 235, 19). We have hypothesized that CP27 exhibits its effects on organogenesis by affecting individual cell function. Based on the CP27 expression pattern we have selected the CP27 expressing embryonic fibroblast cell line BALB/c 3T3 to determine the effects of CP27 on cell function. CP27 loss of function strategies were performed by adding 5, 12.5 or 25 micro g/ml anti-CP27 antibody to cultured BALB/c 3T3 cells and comparing the results to controls in which identical concentrations of rabbit serum were added to the culture medium. Other controls included an antibody against another extracellular matrix protein amelogenin (negative control) and anti-CP27 antibodies directed against other areas of the CP27 molecule (positive control). Following cell culture, cell viability, apoptosis, cell proliferation, cell shape, cellular attachment and fibronectin matrix production were assayed using MTT colourimetric assay, BrdU staining, morphometry, immunostaining and western blot analysis. Block of CP27 function using an antibody strategy resulted in the following significant changes: (i) reduced viability, (ii) increased number of apoptotic cells, (iii) reduced proliferation, (iv) alterations in cell shape, (v) loss of attachment, and (vi) reduction in fibronectin matrix production. There was also a redistribution in fibronectin matrix organization demonstrated by immunohistochemistry. We conclude that CP27 plays an important role in the maintance of normal cell function and that CP27 block leads to significant changes in cellular behaviour.     

Crystal structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain

Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain of Caenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three beta-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.     

Processing of viral envelope glycoprotein by the endomannosidase pathway: evaluation of host cell specificity

Endo-alpha-D-mannosidase is an enzyme involved in N-linked oligosaccharide processing which through its capacity to cleave the internal linkage between the glucose-substituted mannose and the remainder of the polymannose carbohydrate unit can provide an alternate pathway for achieving deglucosylation and thereby make possible the continued formation of complex oligosaccharides during a glucosidase blockade. In view of the important role which has been attributed to glucose on nascent glycoproteins as a regulator of a number of biological events, we chose to further define the in vivo action of endomannosidase by focusing on the well characterized VSV envelope glycoprotein (G protein) which can be formed by the large array of cell lines susceptible to infection by this pathogen. Through an assessment of the extent to which the G protein was converted to an endo-beta-N- acetylglucosaminidase (endo H)-resistant form during a castanospermine imposed glucosidase blockade, we found that utilization of the endomannosidase-mediated deglucosylation route was clearly host cell specific, ranging from greater than 90% in HepG2 and PtK1 cells to complete absence in CHO, MDCK, and MDBK cells, with intermediate values in BHK, BW5147.3, LLC-PK1, BRL, and NRK cell lines. In some of the latter group the electrophoretic pattern after endo H treatment suggested that only one of the two N-linked oligosaccharides of the G protein was processed by endomannosidase. In the presence of the specific endomannosidase inhibitor, Glcalpha1-->3(1- deoxy)mannojirimycin, the conversion of the G protein into an endo H- resistant form was completely arrested. While the lack of G protein processing by CHO cells was consistent with the absence of in vitro measured endomannosidase activity in this cell line, the failure of MDBK and MDCK cells to convert the G protein into an endo H-resistant form was surprising since these cell lines have substantial levels of the enzyme. Similarly, we observed that influenza virus hemagglutinin was not processed in castanospermine-treated MDCK cells. Our findings suggest that studies which rely on glucosidase inhibition to explore the function of glucose in controlling such critical biological phenomena as intracellular movement or quality control should be carried out in cell lines in which the glycoprotein under study is not a substrate for endomannosidase action.      

储藏中药材孳生粉螨的调查

采用清水漂浮法和塔氏电热集螨器分离法,共分离中药材样品129种,1290份,从中分离出粉螨44种,隶属7科25属。得出结论:中药材粉螨的污染严重,应加强对中药材螨类的防治,以保护中药材及预防人体螨病。