Biomedical subjects

Yong Yu

Publications and source records attributed to Yong Yu.

32 records · Page 2Linked to original sources

PubMed2005

Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein.

Streptavidin, a homotetrameric protein with extremely tight biotin binding (K(d) < or = 10(-14) M), has been widely used as an affinity reagent. Its utility would be increased by engineering single-chain mutants with a wide spectrum of affinities, more suitable for phage-display and chip technologies. By a circular permutation procedure, we converted streptavidin to a single-chain dimer (SCD) with two biotin-binding sites and introduced random mutations by error-prone PCR. Clones from a phagemid library, expressed as gene-3 fusion proteins on M13 bacteriophage, were panned with biotinylated beads, and SCD genes from affinity-enriched phage were subcloned to produce soluble proteins. Purification of products from the original gene and two mutants by FPLC and analysis by MALDI-TOF MS showed they exist in both dimeric (single-chain) and tetrameric (two-chain) forms, which were further characterized for their binding affinity to biotin-4-fluorescein (B4F) by fluorescence polarization and intensity measurements. K'(d) values for B4F ranged from approximately 10(-11) to 10(-10) M, although K(d) values for biotin ranged from 10(-6) to 10(-5) M. These results point to the possibility of combining an SCD streptavidin mutant with B4F derivatives to create a fluorescence-tagged affinity system with tight but still-reversible interaction that could be used sequentially with ordinary streptavidin-biotin for composite separation or analysis steps.

Affinity Labels↗

PubMed2005

Influence of electrostatic interaction on fibrinogen adsorption on gold studied by imaging ellipsometry combined with electrochemical methods.

Imaging ellipsometry was combined with electrochemical methods for studying electrostatic interactions of protein and solid surfaces. The potential of zero charge for gold-coated silicon wafer/solution interfaces wad determined by AC impedance method. The potential of the gold-coated silicon wafer was controlled at the potential of zero charge, and the adsorption of fibrinogen on the potential-controlled and non-controlled surfaces was measured in real time at the same time by imaging ellipsometry. The effect of electrostatic interaction was studied by comparing the difference between the potential of controlled adsorption and the potential of noncontrolled adsorption. It was shown that the rate of fibrinogen adsorption on the potentiostatic surface was faster than that on the nonpotentiostatic surface. The electrostatic influence on fibrinogen adsorption on the gold-coated silicon wafer was weak, so the hydrophobic interaction should be the major affinity.

Adsorption↗

PubMed2005

An avidin-like domain that does not bind biotin is adopted for oligomerization by the extracellular mosaic protein fibropellin.

The protein avidin found in egg white seems optimized for binding the small vitamin biotin as a stable homotetramer. Indeed, along with its streptavidin ortholog in the bacterium Streptomyces avidinii, this protein shows the strongest known noncovalent bond of a protein with a small ligand. A third known member of the avidin family, as similar to avidin as is streptavidin, is found at the C-terminal ends of the multidomain fibropellin proteins found in sea urchin. The fibropellins form a layer known as the apical lamina that surrounds the sea urchin embryo throughout development. Based upon the structure of avidin, we deduced a structural model for the avidin-like domain of the fibropellins and found that computational modeling predicts a lack of biotin binding and the preservation of tetramerization. To test this prediction we expressed and purified the fibropellin avidin-like domain and found it indeed to be a homotetramer incapable of binding biotin. Several lines of evidence suggest that the avidin-like domain causes the entire fibropellin protein to tetramerize. We suggest that the presence of the avidin-like domain serves a structural (tetrameric form) rather than functional (biotin-binding) role and may therefore be a molecular instance of exaptation-the modification of an existing function toward a new function. Finally, based upon the oligomerization of the avidin-like domain, we propose a model for the overall structure of the apical lamina.

Amino Acid Sequence↗

PubMed2004

pH-induced conformational changes in the soluble manganese-stabilizing protein of photosystem II.

In this paper, we analyzed the pH-induced changes in the conformational states of the manganese-stabilizing protein (MSP) of photosystem II. Distinct conformational states of MSP were identified using fluorescence spectra, far-UV circular dichroism, and pressure-induced unfolding at varying suspension pH values, and four different conformational states of MSP were clearly distinguished using the center of fluorescence spectra mass when suspension pH was altered from 2 to 12. MSP was completely unfolded at a suspension pH above 11 and partly unfolded below a pH of 3. Analysis of the center of fluorescence spectral mass showed that the MSP structure appears stably folded around pH 6 and 4. The conformational state of MSP at pH 4 seems more stable than that at pH 6. Studies of peak positions of tryptophan fluorescence and MSP-bound 1-anilinonaphthalene-8-sulfonic acid fluorescence spectra supported this observation. A decrease in the suspension pH to 2 resulted in significant alterations in the MSP structure possibly because of protonation of unprotonated residues at lower pH, suggesting the existence of a large number of unprotonated amino acid residues at neutral pH possibly useful for proton transport in oxygen evolution. The acidic pH-induced conformational changes of MSP were reversible upon increase of pH to neutral pH; however, N-bromosuccinimide modification of tryptophan (Trp241) blocks the recovery of pH-induced conformational changes in MSP, implying that Trp241 is a key residue for the unfolded protein to form a functional structure. Thus, pH-induced structural changes of stable MSP (pH 6-4) may be utilized to analyze its functionality as a cofactor for oxygen evolution.

Bromosuccinimide↗

PubMed2004

A grey multi-objective programming approach for sustainable land-use in the Miyun Reservoir basin, China.

Miyun Reservoir is the most important water source to Beijing City. Land-use of the basin plays a great role in the protection of water resources. Hence a sustainable land-use planning is required to optimize land-use structure and protect water resources in the basin. Based on the complete land-use system analysis in Miyun, a grey multi-objective programming to basin land-use(GMOPBLU) model was developed and applied to land-use planning. Two alternatives were produced and analyzed by means of interactive adjustment and scenario analysis. The results showed the GMOPBLU model is a valuable approach for basin land-use planning.

China↗

PubMed2003

Study of the adsorption of fibrinogen on gold-coated silicon wafer by an impedance method.

In 0.1 mol/l KH(2)PO(4)-Na(2)HPO(4) (pH 7.80) buffer solution, the potential of zero charge (PZC) and the open circuit potential of gold-coated silicon were determined to be about -0.6 and +0.10 V (vs SCE), respectively. The open circuit potential was higher than the PZC, which indicated that the surface of the gold-coated electrode had a positive charge. The ellipsometry experiment showed that the adsorption of fibrinogen onto the gold-coated silicon wafer surface arrived at a saturated state when the adsorption time exceeded 50 min. The percentage of surface without adsorbed protein, theta, was about 63%. This means that the proportion of surface actually occupied by fibrinogen was only about 37% after the adsorption arrived at saturation. The solution/protein capacitance value was determined in an impulse state around -0.59 V (vs SCE) and was stable (4.2x10(-5) F) at other potentials.

Adsorption↗

PubMed2003

[The clinical significance of the expression of four cell cycle regulators in breast papillomotosis].

OBJECTIVE: To investigate the clinical significance of the protein expression of 4 cell cycle regulators in papillomatosis of the breast. METHODS: Immunohistochemistry was used to examine the protein expression of CyclinD1, p16, E2F-1 and Rb in 4 groups (196 cases) with mild papillomatosis (MP), moderate papillomatosis (MoP), serious papillomatosis (SP) and ductal carcinoma in situ (DCIS). RESULTS: There were significant difference of the positive rate and the intensity of CyclinD1, p16, E2F-1 and Rb expression among the groups, respectively (chi(2) were 16.702, 20.742, 40.335, 42.317; 19.120, 29.469, 45.080, 46.920, P < 0.01. Meaning time there were significant difference in the expression of these 4 factors between MoP and SP (P < 0.05). There was significance of E2F-1 or Rb expression between SP and DCIS (P < 0.01), but there was no significance to be found in the expression of CyclinD1 or p16 between the two groups (P > 0.05). Rb was screened as the highest risk factor by Odd Risk Logistic Analysis. CONCLUSION: CyclinD1, p16, E2F-1 and Rb played the important roles from MP into SP and then DCIS. E2F-1 and Rb can be used as the assistant indicators on the differentiation of SP and DCIS. Rb may be helpful in screening high risk cases from SP or MoP.

Breast Neoplasms↗

PubMed2003

Structural effects of quinacrine binding in the open channel of the acetylcholine receptor.

Noncompetitive inhibitors of the nicotinic acetylcholine (ACh) receptors suppress cation flux directly by binding in and blocking the open channel or indirectly by stabilizing closed states of the receptor. The lidocaine derivative QX-314 and the acridine derivative quinacrine act directly as open channel blockers, but can act indirectly as well. The binding site for quinacrine in the open channel of mouse-muscle ACh receptor was mapped in cysteine-substituted mutants of the alpha subunit expressed with wild-type beta, gamma, and delta subunits. In the open state, substituted cysteines in the inner half of the second membrane-spanning segment (M2), but not in the outer half, were protected by quinacrine from reaction with 2-aminoethyl methanethiosulfonate. In addition, an alkylating derivative, quinacrine mustard, affinity labeled a subset of the substituted cysteines in M2, but only in the open state. These results, mapped onto a model of the open channel surrounded by five alpha-helical M2s, imply that quinacrine binds midway down M2 in the same site previously mapped for QX-314. A cysteine substituted for a residue in the outer third of alphaM1, which reacted with 2-aminoethyl methanethiosulfonate only in the presence of ACh, reacted faster in the additional presence of quinacrine or QX-314. It is proposed that channel opening involves both the opening of the resting gate at the inner end of M2 and the removal of an obstruction formed by the outer end of M1 that retards diffusion of blockers into the closed channel. Blocker binding in the open channel causes a further change in structure.

Amino Acid Substitution↗

PubMed2003

Ets1 as a marker of malignant potential in gastric carcinoma.

AIM: Ets1 proto-oncogene is a transcription factor involved in the activation of several genes of tumor invasion and metastasis. We aimed to determine the relationship between the extent and intensity of Ets1 expression and patients' clinicopathological factors in gastric carcinoma. METHODS: Immunohistochemical analysis was performed for gastric tumor paraffin-embedded sections, followed by image analysis. RESULTS: Ets1 was not expressed in the normal gastric epithelium and its surrounding cells. The percentage of Ets1 expressing cells detected increased significantly in both epithelial tumor and stromal cells from high T classification, lymph node metastasis positive, clinical advanced-stage groups (P<0.001). The level of Ets1 staining in epithelial tumor cells also reflected the degree of cell differentiation. The percentage of epithelial and stromal cells expressing Ets1 was significantly correlated with the presence of lymph node metastasis (P=0.014 and P<0.001 respectively). Ets1 expression was not observed in tissue samples from patients with benign gastric ulcers. CONCLUSION: Ets1 protein expression in epithelial tumor cells reflects the degree of differentiation, and the percentage of Ets1 positive tumor and stromal cells correlates with lymph node metastasis. Thus Ets1 is a valuable marker of malignant potential in terms of invasiveness and metastasis of gastric carcinoma. It is also possible that inhibition of Ets1 is a potential avenue for therapy in gastric cancer.

Adenocarcinoma↗

PubMed2003

[Early fracture fixation alleviates gut barrier function damage after multiple firearm injuries in pigs].

OBJECTIVE: To explore if early fracture fixation can alleviate gut barrier function damage caused by multiple firearm injuries in pigs. METHODS: Twelve healthy pigs were subjected to tangential fracture of parietal bone and comminuted fractures of bilateral femora (ISS >or= 16) due to 5.8 mm bullets shooting and these pigs were divided randomly into 2 groups. Control group (n = 6) were not treated at all. Fracture fixation Group (n = 6) were managed by immediate fracture fixation of bilateral femora with intramedullary nails. Plasma concentration of D-lactate, DAO and endotoxin (in portal vein) were detected at different intervals before and after trauma. The portal vein blood was cultured and the percentage of positive isolation was calculated. The concentration of DAO in small bowel was also detected 72 hours later after trauma. RESULTS: In control group, the plasma concentrations of D-lactate, DAO and endotoxin increased at early stage and kept high till 72 hours after trauma; the percentage of positive blood culture was 63.3%. In Group F, the levels of plasma D-lactate, DAO and endotoxin were also elevated at early stage (6 - 12 h), but declined significantly from 24 h or 48 h after trauma compared with control group (P < 0.05), and the percentage of positive blood culture was lower (30.0%, P < 0.05). The concentrations of DAO in small bowel decreased in both groups, but to a less extent in Group F. CONCLUSION: Bacterial and endotoxin translocation emerged with increasing gut permeability after multiple firearm injuries. The damage of gut barrier function could be alleviated and the chance of enterogenous infection could be by early fracture fixation after trauma.

Animals↗

PubMed2002

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II.

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.

Molecular Weight↗

PubMed2002

[The relationship between use of the antibiotics in a burn unit and the change of the drug-resistance of Staphylococcus aureus].

OBJECTIVE: To investigate the relationship between the use of antibiotics in a burn unit and the change in the drug - resistance of Staphylococcus aureus (S. aureus). METHODS: By calculating the defined daily doses (DDD) of accumulated consumption of antibiotics per unit time and expense consumption, the use of different kinds of antibiotics in a burn unit in recent five years was analyzed, and correlation analysis between the change of antibiotics consumption amount and the change in drug - resistance level of S. aureus were carried out. RESULTS: Amikacin, gentamycin and cephazolin were the commonest antibiotics used in our burn unit. They were relatively cheaper than some other antibiotics. The consumption amount of compound antibiotics application was negatively correlated with the penicillin resistance level of S. aureus. Seven correlation coefficients between the consumption of first generation cephalosporins and seven coefficiences of resistance rate of S. aureus were negative. The consumption amount of the 3rd generation of cephalosporin application was positively related to the resistance of S. aureus to erythromycin and oxacillin. CONCLUSION: Accumulated DDD might be one of the ideal indices of reflecting antibiotic use. The changes in the consumption amount of the 1st and 3rd generation of cephalosporins containing beta-lactamase-inhibitor might affect the drug-resistance levels of S. aureus to some degree.

Anti-Bacterial Agents↗

PubMed1999

The Influence of NBS Modification of 241Trp on the Reconstitution of 33 kD Protein with PS and on the Recovery of Oxygen-evolving Activity.

The extrinsic 33 kD protein of photosystemII(PSII) plays an important role in the stabilizing of manganese cluster and maintaining high oxygen-evolving activity of PSII. In this research, (241)Trp, the only tryptophan in the 33 kD protein, was modified by N-Bromosuccinimide. The pH-dependence of modification suggests that this tryptophan is buried in the hydrophobic interior of the protein. The protein's capability of reconstitution to the PSII ecreased after modification, and no oxygen-evolving activity of PS was recovered after the reconstitution. Results suggest that (241)Trp of the 33 kD protein is essential for the binding of the protein to the PS and the normal oxygen-evolving activity.

Journal Article↗

PubMedDate not supplied

Native disulfide bonds in plasma retinol-binding protein are not essential for all-trans-retinol-binding activity.

A human plasma retinol-binding protein (RBP) mutant, named RBP-S, has been designed and produced in which the six native cysteine residues, involved in the formation of three disulfide bonds, have been replaced with serine. A hexa-histidine tag was also added to the C-terminus of RBP for ease of purification. The removal of the disulfide bonds led to a decrease in the affinity of RBP for all trans-retinol. Data indicates all-trans-retinol binds RBP and RBP-S with Kd = 4 x 10(-8) M and 1 x 10(-7) M, respectively, at approximately 20 degrees C. RBP-S has reduced stability as compared to natural RBP below pH 8.0 and at room temperature. Circular dichroism in the far-UV shows that there is a relaxation of the RBP structure upon the removal of its disulfide bonds. Circular dichroism in the near-UV shows that in the absence of the disulfide bonds, the optical activity of RBP is higher in the 310-330 nm than in the 280-290 nm range. This work suggests that the three native disulfide bonds aid in the folding of RBP but are not essential to produce a soluble, active protein.

Circular Dichroism↗