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Yi Lu

Publications and source records attributed to Yi Lu.

At least 127 records · Page 7Linked to original sources
PubMed2003

A lead-dependent DNAzyme with a two-step mechanism.

A detailed biochemical and mechanistic study of in vitro selected variants of 8-17 DNAzymes is presented. Even though the 8-17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg(2+) (called 8-17), 0.5 mM Mg(2+)/50 mM histidine (called Mg5), or 100 microM Zn(2+) (called 17E), all variants are shown to be the most active with Pb(2+) (8-17: k(obs) approximately 0.5 min(-1); Mg5: k(obs) approximately 2 min(-1); 17E: k(obs) approximately 1 min(-1) with 200 microM Pb(2+) at pH 5.0). For the 17E variant of the 8-17 DNAzyme, the single-turnover rate constants followed the order of Pb(2+) >> Zn(2+) >> Mn(2+) approximately Co(2+) > Ni(2+) > Mg(2+) approximately Ca(2+) > Sr(2+) approximately Ba(2+). The catalytic rate is half-maximal at 13.5 microM Pb(2+), 0.97 mM Zn(2+), or 10.5 mM Mg(2+), suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb(2+) > Zn(2+) > Mg(2+). The Pb(2+)-dependent activity increases linearly with pH and the slope of the plot of log k(obs) versus pH is approximately 1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G*T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg(2+) and Zn(2+) catalyze only a transesterification reaction with formation of a product containing a 2',3'-cyclic phosphate, Pb(2+) catalyzes a transesterification reaction followed by hydrolysis of the 2',3'-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8-17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb(2+)-binding sites and their roles in the two-step mechanism.

Base Composition↗
PubMed2003

A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles.

A highly sensitive and selective colorimetric lead biosensor based on DNAzyme-directed assembly of gold nanoparticles is reported. It consists of a DNAzyme and its substrate that can hybridize to a 5'-thio-modified DNA attached to gold nanoparticles. The hybridization brings gold nanoparticles together, resulting in a blue-colored nanoparticle assembly. In the presence of lead, the DNAzyme catalyzes specific hydrolytic cleavage, which prevents the formation of the nanoparticle assembly, resulting in red-colored individual nanoparticles. The detection level can be tuned to several orders of magnitude, from 100 nM to over 200 muM, through addition of an inactive variant of the DNAzyme. The concept developed here can be applied to the design of nucleic acid enzyme/nanoparticle sensors for analytes that are subject to in vitro selection, and thus can significantly expand the scope of nanomaterial applications and provide a novel approach to designing simple colorimetric biosensors.

Biosensing Techniques↗
PubMed2003

Neutral thiol as a proximal ligand to ferrous heme iron: implications for heme proteins that lose cysteine thiolate ligation on reduction.

Cysteine plays a key role as a metal ligand in metalloproteins. In all well-recognized cases, however, it is the anionic cysteinate that coordinates. Several cysteinate-ligated heme proteins are known, but some fail to retain thiolate ligation in the ferrous state, possibly following protonation to form neutral cysteine. Ligation by cysteine thiol in ferrous heme proteins has not been documented. To establish spectroscopic signatures for such systems, we have prepared five-coordinate adducts of the ferrous myoglobin H94G cavity mutant with neutral thiol and thioether sulfur donors as well as six-coordinate derivatives such as with CO and, when possible, with NO and O(2). A thiol-ligated oxyferrous complex is reported, to our knowledge for the first time. Further, a bis-thioether ferrous H93G model for bis-methionine ligation, as found in Pseudomonas aeruginosa bacterioferritin heme protein, is described. Magnetic CD spectroscopy has been used due to its established ability in axial ligand identification. The magnetic CD spectra of the H93G complexes have been compared with those of ferrous H175CD235L cytochrome c peroxidase to show that its proximal ligand is neutral cysteine. We had previously reported this cytochrome c peroxidase mutant to be cysteinate-ligated in the ferric state, but the ferrous ligand was undetermined. The spectral properties of ferrous liver microsomal cytochrome P420 (inactive P450) are also consistent with thiol ligation. This study establishes that neutral cysteine can serve as a ligand in ferrous heme iron proteins, and that ferric cysteinate-ligated heme proteins that fail to retain such ligation on reduction may simply be ligated by neutral cysteine.

Circular Dichroism↗
PubMed2003

The role of copper and protons in heme-copper oxidases: kinetic study of an engineered heme-copper center in myoglobin.

To probe the role of copper and protons in heme-copper oxidase (HCO), we have performed kinetic studies on an engineered heme-copper center in sperm whale myoglobin (Leu-29 --> HisPhe-43 --> His, called Cu(B)Mb) that closely mimics the heme-copper center in HCO. In the absence of metal ions, the engineered Cu(B) center in Cu(B)Mb decreases the O(2) binding affinity of the heme. However, addition of Ag(I), a redox-inactive mimic of Cu(I), increases the O(2)-binding affinity. More importantly, copper ion in the Cu(B) center is essential for O(2) reduction, as no O(2) reduction can be observed in copper-free, Zn(II), or Ag(I) derivatives of Cu(B)Mb. Instead of producing a ferryl-heme as in HCO, the Cu(B)Mb generates verdoheme because the engineered Cu(B)Mb may lack a hydrogen bonding network that delivers protons to promote the heterolytic OO cleavage necessary for the formation of ferryl-heme. Reaction of oxidized Cu(B)Mb with H(2)O(2), a species equivalent in oxidation state to 2e(-), reduced O(2) but, possessing the extra protons, resulted in ferryl-heme formation, as in HCO. The results showed that the Cu(B) center plays a critical role in O(2) binding and reduction, and that proton delivery during the O(2) reduction is important to avoid heme degradation and to promote the HCO reaction.

Amino Acid Sequence↗
PubMed2003

New highly sensitive and selective catalytic DNA biosensors for metal ions.

While remarkable progress has been made in developing sensors for metal ions such as Ca(II) and Zn(II), designing and synthesizing sensitive and selective metal ion sensors remains a significant challenge. Perhaps the biggest challenge is the design and synthesis of a sensor capable of specific and strong metal binding. Since our knowledge about the construction of metal-binding sites in general is limited, searching for sensors in a combinatorial way is of significant value. Therefore, we have been able to use a combinatorial method called in vitro selection to obtain catalytic DNA that can bind a metal ion of choice strongly and specifically. The metal ion selectivity of the catalytic DNA was further improved using a 'negative selection' strategy where catalytic DNA that are selective for competing metal ions are discarded in the in vitro selection processes. By labeling the resulting catalytic DNA with a fluorophore/quencher pair, we have made a new class of metal ion fluorescent sensors that are the first examples of catalytic DNA biosensors for metal ions. The sensors combine the high selectivity of catalytic DNA with the high sensitivity of fluorescent detection, and can be applied to the quantitative detection of metal ions over a wide concentration range and with high selectivity. The use of DNA sensors in detection and quantification of lead ions in environmental samples such as water from Lake Michigan has been demonstrated. DNA is stable, cost-effective, environmentally benign, and easily adaptable to optical fiber and microarray technology for device manufacture. Thus, the DNA sensors explained here hold great promise for on-site and real-time monitoring of metal ions in the fields of environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial process monitoring.

Biosensing Techniques↗
PubMed2003

Effects of long-term tea polyphenols consumption on hepatic microsomal drug-metabolizing enzymes and liver function in Wistar rats.

AIM: To investigate the effects of long-term tea polyphenols (TPs) consumption on hepatic microsomal drug-metabolizing enzymes and liver function in rats. METHODS: TPs were administered intragastrically to rats at the doses of 833 mg.kg(-1).d(-1) (n=20) and 83.3 mg.kg(-1).d(-1) (n=20) respectively for six months. Controlled group (n=20) was given same volume of saline solution. Then the contents of cytochrome P450, b5, enzyme activities of aminopyrine N-demethylase (ADM), glutathione S-transferase (GST) and the biochemical liver function of serum were determined. RESULTS: The contents of cytochrome P450 and b5 in the livers of male rats in high dose groups (respectively 2.66 +/- 0.55, 10.43 +/- 2.78 nmol.mg MS pro(-1)) were significantly increased compared with the control group (1.08 +/- 1.04, 5.51 +/- 2.98 nmol.mg MS pro(-1); P<0.01, respectively). The enzymatic activities of ADM in the livers of female rats in high dose groups (0.91 +/- 0.08 mmol.mg MS pro(-1)min(-1)) were increased compared with the control group (0.82 +/- 0.08 mmol.mg MS pro(-1).min(-1); P<0.05). The GST activity was unchanged in all treated groups, and the function of liver was not obviously changed. CONCLUSION: The antidotal capability of rats' livers can be significantly improved after long-term consumption of TPs. There are differences in changes of drug-metabolizing enzymes between the sexes induced by TPs and normal condition.

Aminopyrine N-Demethylase↗
PubMed2003

Cloning and expression of ornithine decarboxylase gene from human colorectal carcinoma.

AIM: To construct and express ODC recombinant gene for further exploring its potential use in early diagnosis of colorectal carcinoma. METHODS: Total RNA was extracted from colon cancer tissues and amplified by reverse-transcription PCR with two primers, which span the whole coding region of ODC. The synthesized ODC cDNA was cloned into vector pQE-30 at restriction sites BamH I and Sal I which constituted recombinant expression plasmid pQE30-ODC. The sequence of inserted fragment was confirmed by DNA sequencing, the fusion protein including 6His-tag was facilitated for purification by Ni-NTA chromatographic column. RESULTS: ODC expression vector was constructed and confirmed with restriction enzyme digestion and subsequent DNA sequencing. The DNA sequence matching on NCBI Blast showed 99 % affinity. The vector was transformed into E. coli M15 and expressed. The expressed ODC protein was verified with Western blotting. CONCLUSION: The ODC prokaryote expression vector is constructed and thus greatly facilitates to study the role of ODC in colorectal carcinoma.

Cloning, Molecular↗
PubMed2003

[Studies of retinal dopamine transporter in experimental myopia].

OBJECTIVE: To investigate the distribution of dopamine transporter (DAT) in the chicken eye and to explore the role of DAT in the occurrence of experimental myopia. METHODS: Thirty 2-day-old chickens were divided into four groups. Chicken eyes were fitted with lenses of -10 D (Group 1), -20 D (Group 2) and translucent goggles (Group 3) unilaterally. Chickens without any treatments were used as the control (Group 4). The refraction and the axial eye length of all chickens were measured after 3 weeks, then, all chickens were given an intramuscular injection of (125)I-beta-CIT [2beta-carbomethoxy-3beta-(4-iodophenyl) tropane] and sacrificed two hours after injection. Retinal pigment epithelium (RPE) and neural retina were dissected from the eye as a whole layer in Groups 1 and 4; and dissected separately in Groups 2 and 3. Radioactive DAT from each specimen was assayed by gamma-counter. RESULTS: In Groups 2 and 3, the radioactive DAT value in the RPE from the experimental eyes was significantly greater than that in the neural retina and also greater than those in the RPE from the control eyes (P < 0.01). In Groups 1, 2, and 3, the radioactive DAT value in the whole retina or RPE from the experimental eyes was significantly greater than those from the control eyes (P < 0.01). CONCLUSIONS: Retinal DAT is located mainly in the RPE and may be involved in the occurrence of both lens-induced myopia and form-deprivation myopia. These methods may provide a new approach for further studying the role of dopamine system in the occurrence of experimental myopia.

Animals↗
PubMed2003

[Platelet phospholipase A2 mRNA content changes and cDNA cloning in rat blood with bacteria infection].

AIM: To explore the changes of rat platelet phospholipase A2 (PLA2) mRNA content in bacteria infected rat and study the cDNA and amino acid sequences of the PLA2 structure to lay a good foundation for the development of new antibiotics. METHODS: The PLA2 mRNA level in blood was determined by RT-PCR. The DNA sequence was cloned and analyzed. RESULTS: After injection of bacteria in rats, the mRNA level of PLA2 in blood increased markedly. The cDNA and amino acid sequence were highly homologous to other PLA2 cDNA from different tissues of the rat. CONCLUSION: Platelet PLA2 in blood responded quickly to bacteria infection in gene level. Therefore, the PLA2 protein was produced increasingly which was shown to control the infection with bacteria. Although there are little difference between PLA2 cDNA cloned from blood and other sources in DNA and amino acid sequences, the catalytic site for enzymatic activity and basic structure are identical.

Amino Acid Sequence↗
PubMed2003

[Research on the X-ray fluorescence spectrometry method to determine trace elements in kimberlite].

It is very important to detect trace elements for kilmberlite. Through improving the working conditions of X-ray fluorescence spectrometer and optimizing the analytical conditions, the determination method of trace elements, such as Sc, Cr, Ni, Y, Nb, La, in kimberlite was worked out. The method has been successfully applied to the determination of trace elements in over 2 thousand samples of kimberlite from Liaoning province. The detection limits of the method were relatively low (the detection limit of Sc droped from 9.54 to 2.83 micrograms.g-1 and the detection limit of La droped from 21.68 micrograms.g-1 to 9.18 micrograms.g-1), i.e. 2.83, 2.15, 2.20, 1.17, 1.05 and 9.18 micrograms.g-1 for Sc, Cr, Ni, Y, Nb and La, respectively. The precision of the method was very high with 2.10%-7.09% of RSD (n = 20). Compared with ICP spectrometry this method is satisfactory. The method has proven to be simple and rapid with low cost and high efficiency.

English Abstract↗
PubMed2003

[Studies of poly(vinyl alcohol)/hydroxylapatite hydrogels compounds for cartilage implantation].

The structures and properties of polyvinyl alcohol (PVA)/hydroxylapatite(HA) composites were investigated. The components and processing conditions were studied for preparation of optimum compound hydrogels with good lubricating feature and high bioactivity. The properties, such as stretch strength, compress strength, friction, compress stress relaxation of various compound hydrogels were compared. The micro-morphology of PVA hydrogels and PVA/HA hydrogels were investigated by use of SEM. It was found that the addition of HA could improve the lubricating feature and mechanical strength of hydrogels, and its compress stress relaxation properties were closer to those of natural cartilages.

Biocompatible Materials↗
PubMed2003

The role of inflammatory mediators in severe acute pancreatitis and regulation of glucocorticoids.

OBJECTIVE: To investigate the effect of glucocorticoids on systemic inflammatory mediator release in rats with acute pancreatitis and the outcome of dexamethasone in treatment of acute pancreatitis. METHODS: Sixty-eight Wistar rats were divided into sham, acute pancreatitis, and treatment (intravenous dexamethasone 0.5 mg/kg) groups. Experimental acute pancreatitis was induced by the injection of 5% sodium taurocholate (0.1 ml/100 mg body weight) into the pancreatic-biliary duct. The blood samples were obtained and examined for 6-keto-PGI1alpha, TXB2 and IL-6 postoperatively at 3, 6 and 12 hours, respectively. The pancreatic samples were evaluated by a blinded method. Twelve-hour survival rate was determined and compared between the groups. RESULTS: The high serum concentrations of 6-keto-PGI1alpha, TXB2 and IL-6 were noted in the rats with acute pancreatitis associated with pancreatic hemorrhage and necrosis. Their 12-hour survival rate was 42.9%. The rats in the treatment group survived with significantly reduced serum concentrations of 6-keto-PGI1alpha, TXB2 and IL-6 (P<0.05). Their pancreatic morphology was normal. CONCLUSION: Dexamethasone may reduce the serum concentration of 6-keto-PGI1alpha, TXB2, and IL-6, and the severity of acute pancreatitis while increasing the survival rate of rats with acute pancreatitis.

6-Ketoprostaglandin F1 alpha↗
PubMed2003

[Carbachol alleviates inflammation in gut tissue during ischemia/reperfusion in rats].

OBJECTIVE: To investigate the effects of carbachol on local inflammation in gut tissue during ischemia/reperfusion (I/R). METHODS: A jejunal sac was formed in Wistar rats. The superior mesenteric artery was occluded (SMAO) for 60 minutes followed by reperfusion for another 60 minutes. Animals were divided into three groups, pretreatment group (carbachol was injected into the jejunal sac 30 minutes after SMAO, 0.1 mg/kg), treatment group (carbachol was injected in same dosage into the jejunal sac 30 minutes after reperfusion), and controls (saline injection). The contents of tumor necrosis factor-alpha(TNF-alpha) and activity of myeloperoxidase (MPO) in gut tissue were determined at 1 hours, 2.5 hours and 6 hours after SMAO. RESULTS: The contents of TNF-alpha and activity of MPO were significantly decreased in pretreatment and treatment groups compared with control group at 2.5 hours after SMAO (both P<0.05). There were no differences in both contents between pretreatment group and treatment group at any specified time. It was also found that there were less inflammatory pathological changes in the gut tissues in the two treated groups than that of control. CONCLUSION: The RESULTS suggest that carbachol could alleviate gut inflammatory response during gut ischemia/reperfusion injury by inhibiting proinflammatory cytokine release.

Animals↗
PubMed2003

[Advances in the study of vincristine: an anticancer ingredient from Catharanthus roseus].

Vincristine is a dimer-indo-alkaloid which is extracted from the leaves of Catharanthus roseus. It is effective to treat acute lymphocytic cell leukemia, Hodgkin disease and non-Hodgkin disease clinically. But the severe side effects, such as neurotoxic and tissue damage, limit its application. In this paper, we summarize physical, chemical, pharmacological and pharmacokinetical properties of VCR and advances in decreasing its side effects. In clinic, association with other medication is adopted. In pharmaceutics, people adopt some new methods and technology such as conjugation with the antibody, encapsulation in liposomes or controlled release films.

Animals↗
PubMed2002

FRET study of a trifluorophore-labeled DNAzyme.

A fluorescence resonance energy transfer (FRET) study of biomolecules typically employs two fluorophores. The increasing number of branches and complexity of biomolecules call for simultaneously monitoring structures and dynamics of several branches in a single system. Furthermore, despite recent studies that show DNAzymes can be a stable and cost-effective alternative to protein and ribozymes for pharmaceutical and biotechnological applications, no FRET study of DNAzymes has been reported. Here, we describe the FRET study of a trifluorophore-labeled "8-17" DNAzyme, in which each of the three branches is labeled with a different fluorophore. From the study, we found that the (ratio)(A) method that has been commonly used in dual-fluorophore-labeled systems is also applicable to trifluorophore-labeled systems. However, while both FRET efficiency and fluorophore-to-fluorophore distance can be used to measure FRET in dual-fluorophore-labeled systems, only the average distance should be used in trifluorophore-labeled systems. The ability to monitor all three branches in a single system allowed us to reveal new metal-ion-dependent conformational changes in the DNAzyme. The trifluorophore-labeled "8-17" DNAzyme has been found to adopt a two-step folding process in the presence of Zn(2+). Each step is induced by one Zn(2+) binding, with apparent dissociation constants of 19 microM and 260 microM for binding the first and second Zn(2+), respectively. The trifluorophore FRET results are verified by a dual-labeled control experiment. The results demonstrated that the trifluorophore-labeled system is simple and yet powerful in studying complicated biomolecular structure and dynamics and is capable of revealing new sophisticated structural changes that may have functional implications.

DNA, Catalytic↗
PubMed2002

An organometallic route to oligonucleotides containing phosphoroselenoate.

Unlike the widespread use of phosphorothioates in nucleic acid chemistry, complementary research on phosphoroselenoates has been severely limited. Previous routes to DNA and RNA that contain phosphoroselenoates employ elemental Se and KSeCN as Se transfer agents, although these reagents suffer from low or unselective reactivity. The metastability of the Pbond;Se bond demands soluble, selective Se transfer reagents. The organometallic reagent (iPrC(5)H(4))(2)TiSe(5) satisfies these criteria, as we demonstrate by the synthesis of phosphoroselenoate derivatives of mono- and oligonucleotides of DNA and a dinucleotide of RNA. The new general method is compatible with high-throughput phosphoramidate oligonucleotide synthesis, which allows for the preparation of site-specifically labeled oligonucleotides. A (31)P NMR spectroscopy study shows that the phosphoroselenoate of (5')-d(GGAATGTC(Se)TGTCG)-(3') selectively binds to soft Cd(2+) ions but not Mg(2+) ions.

Cations, Divalent↗
PubMed2002

New transition-metal-dependent DNAzymes as efficient endonucleases and as selective metal biosensors.

Like proteins and RNA molecules, many DNA molecules have now been shown to catalyze a variety of reactions and are thus called DNAzymes. With limited building blocks, DNAzymes need to recruit other cofactors in order to match other enzymes in terms of reaction diversity and catalytic efficiency. Several unique properties make transition-metal ions an ideal choice of cofactor for DNAzymes. Indeed, new DNAzymes that bind transition-metal ions with high affinity and selectivity have been obtained through the use of a powerful combinatorial biology tool called in vitro selection. This accomplishment now makes it possible to obtain different classes of metallo-DNAzymes in the laboratory within a short period of time. It also offers a rare opportunity to compare and contrast structural and functional properties of metal-binding sites in proteins and in DNAzymes. The resulting transition-metal-dependent DNAzymes have displayed high activity toward cleavage of DNA and RNA and thus hold promise for their biochemical and pharmaceutical applications. Finally, the use of DNAzymes as a new class of highly sensitive and selective biosensors for metal ions has been demonstrated recently.

Journal Article↗