Biomedical subjects

Yi Lu

Publications and source records attributed to Yi Lu.

At least 109 records · Page 6Linked to original sources

PubMed2004

Osteoblasts induce prostate cancer proliferation and PSA expression through interleukin-6-mediated activation of the androgen receptor.

Prostate cancer (CaP) metastases selectively develop in bone as opposed to other sites through unknown mechanisms. Interleukin-6 (IL-6) is considered to contribute to CaP progression and is produced at high levels in osteoblasts. We hypothesized that osteoblast-derived IL-6 in the bone microenvironment contributes to the fertile soil for CaP growth. Accordingly, human CaP cells, LNCaP, C4-2B and VCaP, were treated with conditioned medium (CM) collected from human osteoblast-like HOBIT cells grown in androgen-depleted medium. We found that CM induced proliferation, prostate-specific antigen (PSA) protein and mRNA expression in a dose-dependent manner in these cell lines as determined by ELISA and real-time PCR, respectively. CM also activated the PSA promoter in these cells. Both HOBIT and primary osteoblast (POB) cells produced high levels of IL-6 measured by bioassay. LNCaP, C4-2B and VCaP cells expressed IL-6, but at much lower levels then the HOBIT and POB and they also expressed the IL-6 receptor mRNA, indicating they can respond to IL-6. Anti-IL-6 antibody added to HOBIT or POB CM dose-dependently inhibited the CM-induced cell proliferation and PSA expression in these CaP cell lines. HOBIT CM induced nuclear translocation of the AR and this was inhibited by anti-IL-6 antibody. Additionally, the antiandrogen bicalutamide inhibited HOBIT CM-induced cell proliferation. These results demonstrate that osteoblasts promote CaP growth through IL-6-mediated activation of the AR. Furthermore, these data underscore the importance of cross-talk between tumor and the bone microenvironment in the development of CaP bone metastases.

Androgens↗

PubMed2004

In vivo visualization of aging-associated gene transcription: evidence for free radical theory of aging.

The expression of a variety of proteins is elevated with aging through unknown mechanisms. The free radical theory of aging promulgates that reactive oxygen species (ROS) promote the aging process. However, the mechanisms as to how ROS contribute to the aging process are not clear. We present data here that demonstrate that aging induced ROS promote aging-associated interleukin-6 (IL-6) gene transcription in mice that are transgenic for the murine IL-6 promoter driving a luciferase reporter cDNA. N-acetylcysteine (NAC), an antioxidant, completely reverses the increased endogenous IL-6 promoter activity in the old mice determined by real-time bioluminescence imaging (BLI). We conclude that ability of ROS to act as secondary messengers and induce gene expression may contribute to the aging process.

Acetylcysteine↗

PubMed2004

Spectroscopic evidence for interactions between hexacyanoiron(II/III) and an engineered purple CuA azurin.

Interactions between hexacyanoiron(II/III) and a dinuclear, mixed valence Cu(A) center in engineered Cu(A) azurin have been investigated by UV-visible (UV-vis) and electron paramagnetic resonance (EPR) spectroscopic techniques. Addition of ferricyanide (hexacyanoiron(III)) to the Cu(A) azurin resulted in a new absorption band around 500 nm in the UV-vis and an isotropic line at g = 2.16 in the EPR spectra. Control experiments, including additions of Cu(II)SO(4) or Cu(I)(CH(3)CN)(4)PF(6) to ferricyanide or ferrocyanide, as well as gel filtration purification of the ferricyanide-Cu(A) azurin adduct indicate complex formation between cupric ion and ferrocyanide ion in the protein. Solvent or small molecule accessibility, metal oxidation state and the presence of more than one metal ion are potential factors important for the complex formation. These findings must be taken into consideration when using ferricyanide or ferrocyanide as redox agents for studying Cu(A) centers in proteins.

Journal Article↗

PubMed2004

Colorimetric biosensors based on DNAzyme-assembled gold nanoparticles.

Taking advantage of recent developments in the field of metallic nanoparticle-based colorimetric DNA detection and in the field of in vitro selection of functional DNA/RNA that can recognize a wide range of analytes, we have designed highly sensitive and selective colorimetric biosensors for many analytes of choice. As an example of the sensor design strategy, a highly sensitive and selective colorimetric lead biosensor based on DNAzyme-directed assembly of gold nanoparticles is reviewed. The DNAzyme consists of an enzyme and a substrate strand, which can be used to assemble DNA-functionalized gold nanoparticles. The aggregation brings gold nanoparticles together, resulting in a blue-colored nanoparticle assembly. In the presence of lead, the DNAzyme catalyzes specific hydrolytic cleavage of the substrate strand, which disrupts the formation of the nanoparticle assembly, resulting in red-colored individual nanoparticles. The application of the sensor in lead detection in leaded paint is also demonstrated. In perspective, the use of allosteric DNA/RNAzymes to expand the range of the nanoparticle-based sensor design method is described.

Biosensing Techniques↗

PubMed2004

Novel cellulase profile of Trichoderma reesei strains constructed by cbh1 gene replacement with eg3 gene expression cassette.

To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the P(cbh1)-eg3-T(cbh1) cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbh1 gene was confirmed by PCR, Southern dot blot and Western hybridization analysis in two transformants denoted as L13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbh1 promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential in ustrial applications.

Bacterial Proteins↗

PubMed2004

Synthesis of erythrocyte glutathione in healthy adults consuming the safe amount of dietary protein.

BACKGROUND: The finding that plasma glutathione turnover decreases as dietary protein intake decreases suggests that the safe amount of dietary protein, although sufficient for maintenance of nitrogen balance, may be insufficient for maintenance of cellular glutathione. OBJECTIVE: Our objective was to determine the effect of the safe protein intake on the erythrocyte glutathione synthesis rate and its relation with urinary 5-L-oxoproline excretion. DESIGN: Erythrocyte glutathione synthesis and urinary 5-L-oxoproline excretion were measured in young adults (6 men and 6 women) by using an infusion of [(13)C(2)]glycine on 3 occasions: initially during the subjects' habitual protein intake (1.13 g.kg(-1).d(-1)) and on days 3 and 10 of consumption of a diet providing the safe protein intake (0.75 g.kg(-1).d(-1)). RESULTS: Compared with baseline values, the fractional synthesis rate of erythrocyte glutathione was significantly lower (P < 0.05) on days 3 and 10 of the diet with the safe protein intake. Urinary 5-L-oxoproline excretion increased significantly (P < 0.05) above baseline by the third day of the diet with the safe protein intake and remained elevated. Erythrocyte glutathione concentrations and absolute synthesis rates decreased by day 3 but recovered to baseline values by day 10. Erythrocyte concentrations of cysteine, methionine, and serine remained unchanged, whereas erythrocyte concentrations of glycine, glutamic acid, and glutamine increased significantly by day 10. CONCLUSION: During adaptation to the safe amount of dietary protein, there are changes in the concentration and kinetics of erythrocyte glutathione that suggest a reduced antioxidant capacity and possible increased susceptibility to oxidant stress.

Adaptation, Physiological↗

PubMed2004

[Effects of carbachol injection in intestine on plasma levels of inflammatory medium in rats during gut ischemia/reperfusion].

OBJECTIVE: To investigate the effects of carbachol injection in intestine on plasma levels of tumor necrosis factor-alpha(TNF-alpha), interleukin-10 (IL-10) and cortisol in rats during gut ischemia/reperfusion. METHODS: Wistar rats were anaesthetized with soluble pentobarbitone, and subjected to superior mesenteric artery occlusion (SMAO) for 60 minutes, followed by reperfusion for 60 minutes. Animals were divided into three groups, pretreated group (carbachol injection in intestine at 30 minutes after SMAO; 0.1 mg/kg), treated group (carbachol injection at 30 minutes from onset of reperfusion), and controls (saline injection). Plasma TNF-alpha, IL-10 and cortisol levels were determined at 1.0, 2.5 and 6.0 hours after SMAO. RESULTS: The plasma levels of TNF-alpha significantly decreased in pretreated and treated groups than those in controls after carbachol injection (both P<0.01). However, the levels of IL-10 and cortisol didn't show significant differences among three groups. CONCLUSION: The RESULTS suggest that carbachol can reduce the proinflammatory cytokine release and have a less inhibitory effect on the anti-inflammatory cytokine. It is indicated that carbachol play a potential role in alleviating systemic inflammatory response during splanchnic ischemia/reperfusion injury.

Animals↗

PubMed2004

Hepatic volumetry with PhotoShop in personal computer.

BACKGROUND: Convenient way to clarify liver volume or tumor volume in the liver is eagerly demanded by hepatobiliary surgeons, for so many aspects of clinical work need to know the liver volumetry. At present, some methods have been used to measure the liver volumetry, such as computed tomography (CT) scans, three-dimensional ultrasound volumetric system([1]) and 3-dimensional sonography([2,3]) et al. But enough volumetric information was failed to obtain by surgeons and a new way of measuring the liver volumetry that can be operated by themselves is exigent. Whereas we devise a new method of using PhotoShop in personal computer to measure the liver volumetry. METHODS: A piece of whole CT film was transformed to a high quality digitized image by digital camera or scanner and then the digitized image was conducted as JPEG file into personal computer. The JPEG image file of CT film was opened by PhotoShop. Determining the edge of interested areas, and the data of pixel values of the interested areas divided by 1 cm2 pixel value will produce the actual area with the unit of square centimeter. If section thickness of CT scan is 1 cm, the sum of the areas of the liver or tumor in all sections naturally is the volume of the liver or tumor. RESULTS: Comparison of 10 hepatic volumes gained by this method and those gained by the GE Prospeed CT set showed a good relativity between the two groups. The volumes of three right lobes were calculated by this method before lobectomy and their real volumes were obtained postoperatively by a volumenometer. Their variation was limited to 5%. CONCLUSIONS: Hepatic volume obtained by PhotoShop is reliable. This method can be used to measure hepatic volume perfectly to meet clinical demand, and many parameters such as liver resection rate, graft volume can be achieved. The disadvantage of this method is the step of copying the pixel value from PhotoShop to Microsoft Excel.

Carcinoma, Hepatocellular↗

PubMed2004

[Defensin-5 and Matrilysin mRNA expression in the intestine of scalded rats and its relation to bacterial translocation].

OBJECTIVE: To investigate the effect of burn injury on the expression of rat defensin-5 (RD-5) and Matrilysin mRNA in the intestine of scalded rats and to determine its relation to bacterial translocation. METHODS: Thirty-two Wistar rats were divided randomly into sham injury group (n=8) and burn group (n=24). Rats of the burn group were subjected to 30% total body surface area (TBSA) full-thickness scald injury. At 8, 24 and 72 hours after the injury, the animals (n=8 at each time point) were sacrificed and ileum was sampled for the determination of the expression of RD-5 mRNA and Matrilysin mRNA with reverse transcription-polymerase chain reaction (RT-PCR) technique. Cultures of mesenteric lymph nodes (MLN), liver, spleen and lung were carried out at 24 hours after the injury to assess the rate of bacterial translocation to visceral organs,and the morphological changes in ileal Paneth cells were observed. RESULTS: The expression of RD-5 mRNA was significantly up-regulated at 8 hours when compared with control group. It was down-regulated sharply at 24 hours but was still higher than control group, and at 72 hours it was down-regulated to a level lower than control. On the contrary, the expression of Matrilysin mRNA was up-regulated continuously to 72 hours, and it was significantly higher than control. Bacterial translocation rate was significantly higher in rats of the burn group at 24 hours (58.3%) than in control group (8.3%, P<0.01), but there were no obvious morphological changes in ileal Paneth cells at the same time point. CONCLUSION: The expressions of both RD-5 mRNA and Matrilysin Mrna are up-regulated in the early stage after the injury, but their time phases are different. The phenomenon might imply that there is a host protective response against bacterial translocation.

Animals↗

PubMed2004

[Change in intestinal function in sepsis in rat].

OBJECTIVE: To study the barrier function, absorption, permeability and peristalsis of intestine in sepsis in rats. METHODS: A Wistar rat model of sepsis was reproduced by ischemia/reperfusion (I/R) of the intestine combined with endotoxin challenge. Animal were randomly divided into normal, I/R 1 hours (I/R 1), I/R 2 hours (I/R 2), I/R 4 hours (I/R 4) and I/RL groups. The following parameters were measured in the experiments: (1) diamin oxidase activity (DAO), D-lactate and D-xylose levels in blood using spectrophotometry; (2) transit function of small intestine; (3) pathological examination of small intestine by light microscope. RESULTS: The results showed that plasma DAO activity was increased in I/R 1, I/R 4 and I/RL (all P<0.05), and small intestinal tissue DAO was decreased in I/R 2 and I/RL (both P<0.05). Negative correlations were found between plasma and intestinal DAO (r=-0.909, P<0.001). Plasma D-lactate was elevated significantly in I/R 1, I/R 2, and I/RL (all P<0.05). D-xylose content was increased at I/R1 and I/RL groups (both P<0.05), and it was significantly higher than controls at 3 hours. Similarly, a positive correlation was found between plasma DAO activity and plasma D-lactate level (r=0.559, P<0.05). CONCLUSION: The intestinal barrier function, absorption function, permeability, and transit are impaired after gut ischemia/reperfusion combined with endotoxin challenge.

Amine Oxidase (Copper-Containing)↗

PubMed2004

[Preliminary results of intraocular lens implantation in infants with cataract under two years old].

OBJECTIVE: To explore the feasibility of anterior and posterior capsulorhexis combined with anterior vitrectomy and intraocular lens implantation in infants with cataract under 2 years old. METHODS: Anterior and posterior capsulorhexis combined with anterior vitrectomy and intraocular lens implantation was performed in 22 cases (27 eyes) of infants with cataract under 2 years old. The results during and after the operation were observed. The mean follow-up time was 12 months. RESULTS: All surgeries were performed smoothly. The visual axis remained clear in 26 eyes (96%) after operation, there was white fibrosis and opacification posterior to the intraocular lens in 1 eye and needed re-operation, up drawn pupil in 3 eyes (11%), pupillary capture in 2 eyes (7%) and posterior synechia of the iris in 4 eyes (15%). CONCLUSIONS: Anterior and posterior capsulorhexis combined with anterior vitrectomy and intraocular lens implantation in infants with cataract under 2 years old can keep visual axis clear and restore the normal anatomy of anterior segment of the eye, the complications are reduced greatly. However, the ultimate visual acuity needs further observation.

Anterior Eye Segment↗

PubMed2004

[Black diaphragm intraocular lens for congenital aniridia or traumatic iris deficiency].

OBJECTIVE: To evaluate the efficacy of a black diaphragm intraocular lens (IOL) implantation for congenital aniridia or traumatic iris deficiency and to explore the mechanism of postoperative complications. METHODS: The therapeutic effects and postoperative complications of twenty-five cases (27 eyes) of black diaphragm intraocular lens implantation were analyzed. Ultrasound biomicroscope (UBM) and gonioscopy were used to observe the position of the IOL haptics and anterior chamber angle structure in patient who developed secondary glaucoma postoperatively. RESULTS: After 3-18 months of follow-up, photophobia was reduced or disappear. The best corrected visual acuity (BCVA) Postoperation was improved 2 lines in 20 eyes, no change or within 1 line in 5 eyes, decreased 2 lines or more in 2 eyes. The complications included secondary glaucoma (10 eyes, 37.0%), astigmatism (17 eyes, 63.0%), corneal decompensation (2 eyes, 7.4%), hyphema (2 eyes, 7.4%), vitreous hemorrhage (3 eyes, 11.1%), and retinal detachment (1 eye, 3.7%). Of the 20 haptics in 10 patients, 9 were adequately located in the sulcus region, the others were located in chamber angle, ciliary processes or posterior to the ciliary processes. The changes of chamber angle included angle recession, anterior adhesion of iris and angle closure. CONCLUSIONS: The black diaphragm intraocular lens implantation provides an effective method in the treatment of congenital aniridia or traumatic iris deficiency. Chamber angle impair is major cause of postoperative secondary glaucoma. Abnormality of lens haptics position, hemorrhage and persistent inflammation were also play a role in secondary glaucoma.

Adolescent↗

PubMed2004

Lamivudine in prevention and treatment of recurrent HBV after liver transplantation.

BACKGROUND: Chronic hepatitis B virus infection is one of the major causes of liver cirrhosis worldwide, especially in Asia. Liver transplantation for the end-stage liver disease with hepatitis B virus(HBV) is commonly complicated by the recurrence of HBV. The present study was designed to evaluate lamivudine in the prevention and treatment of recurrent HBV after liver transplantation. METHODS: Seventeen patients with HBV-related liver disease in a total of 41 patients have received liver transplantation at our hospital since 2001. All the patients were HBV positive before transplantation, 5 of them had acute liver failure. Artificial liver was used in 4 patients with acute liver failure. All of the patients received lamivudine at a dose of 100 mg/d after liver transplantation. Lamivudine and HBIg therapy were given to 3 patients. RESULTS: Liver transplantation was successfully performed in all 17 patients. Three patients died of complications 3-6 months after the transplantation. One patient withdraw from lamivudine therapy and died of liver failure at 14 months after the transplantation. Thirteen patients were followed up from 6 to 18 months. Two viremic patients had HBV recurrence shortly after the transplantation. Two viremic patients who had received HBIg and lamivudine after the transplantation had no evidence of HBV recurrence. CONCLUSIONS: Lamivudine therapy is effective in preventing HBV recurrence after liver transplantation. The recurrence of HBV is closely related to HBV DNA status before liver transplantation.

Adult↗

PubMed2003

Improving fluorescent DNAzyme biosensors by combining inter- and intramolecular quenchers.

A previously reported DNAzyme-based biosensor for Pb(2+) has shown high sensitivity and selectivity at 4 degrees C. In the system, the substrate and the enzyme strand of the DNAzyme are labeled with a fluorophore and a quencher, respectively. In the presence of Pb(2+), the substrate strand is cleaved by the enzyme strand, and the release of the cleaved fragment results in significant fluorescence increase. However, the performance of the sensor decreases considerably if the temperature is raised to room temperature because of high background fluorescence. A careful analysis of the sensor system, including measurement of the melting curve and fluorescence resonance energy-transfer (FRET) study of the free substrate, suggests that a fraction of the fluorophore-labeled substrate strand is dissociated from the enzyme strand, resulting in elevated background fluorescence signals at room temperature. To overcome this problem, we designed a new sensor system by introducing both inter- and intramolecular quenchers. The design was aided by the FRET study that showed the dissociated substrate maintained a random coil conformation with an end-to-end distance of approximately 39 A, which is much shorter than that of the fully extended DNA. With this new design, the background fluorescence was significantly suppressed, with 660% increase of fluorescence intensity as compared to 60% increase for the previous design. This suppression of background fluorescence signals was achieved without losing selectivity of the sensor. The new design makes it possible to use the sensor for practical applications in a wide temperature range. The design principle presented here should be applicable to other nucleic acid-based biosensors to decrease background fluorescence.

Biosensing Techniques↗

PubMed2003

Soluble receptor activator of nuclear factor kappaB Fc diminishes prostate cancer progression in bone.

Prostate cancer (CaP) develops metastatic bone lesions that consist of a mixture of osteosclerosis and osteolysis. We have previously demonstrated that targeting receptor activator of nuclear factor kappaB ligand (RANKL) with osteoprotegerin (OPG) prevents the osteolytic activity of CaP and its ability to establish tumor in bone. However, OPG can block tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, suggesting that the clinical use of OPG may prevent apoptosis of tumors mediated by TRAIL. Thus, methods to block RANKL activity, other than OPG, may be important. Accordingly, we evaluated the ability of soluble murine RANK-Fc (sRANK-Fc) to prevent progression of established CaP in a severe combined immunodeficient mouse implanted with fetal human bone. We first confirmed that sRANK did not block TRAIL-mediated apoptosis of LuCaP cells in vitro and that it did block LuCaP-conditioned media-induced osteoclastogenesis in vitro. Then, LuCaP 35 CaP cells were injected into the marrow space of the bone implanted in the severe combined immunodeficient mice implanted with fetal human bone and allowed to develop into tumors for 6 weeks. Either vehicle or sRANK-Fc was then administered for 6 weeks. sRANK-Fc diminished tumor-induced osteoblastic lesions as demonstrated by radiograph, bone mineral density measurement, and bone histomorphometry. sRANK-Fc also reduced systemic bone remodeling markers, including serum osteocalcin and bone-specific alkaline phosphatase and urine N-telopeptide of collagen. Finally, sRANK-Fc decreased serum prostate-specific antigen levels and tumor volume in the bone, which indicates decreased tumor burden. In contrast, sRANK-Fc had no effect on s.c. implanted LuCaP cells. We conclude that sRANK-Fc is an effective inhibitor of RANKL that diminishes progression of CaP growth in bone through inhibition of bone remodeling.

Animals↗

PubMed2003

Separation of retinoid-induced epidermal and dermal thickening from skin irritation.

The ability of the synthetic retinoid MDI-301, in which the carboxylic acid of 9- cis-retinoic acid (9-cis-RA) is replaced with an ester linkage, to induce epidermal and dermal thickening and skin irritation (erythema and flaking) in hairless (rhino) mice following its topical application was investigated in comparison with that of 14-all- trans-retinoic acid (14-all-trans-RA) and 9-cis-RA. MDI-301 induced epidermal proliferation leading to a thickened epidermis. Treated animals also demonstrated a prominent band of organized connective tissue immediately below the epidermis. In its ability to induce epidermal thickening, MDI-301 was quantitatively similar to 14-all-trans-RA and 9-cis-RA. However, unlike 14-all-trans-RA and 9-cis-RA, which produced skin irritation associated with a perivascular influx of mononuclear leukocytes into the dermis, there was no evidence of irritation with MDI-301 and little leukocyte infiltration. Intraperitoneal injection of either 14-all-trans-RA or MDI-301 also resulted in epidermal and dermal thickening. Irritation of skin was not observed in these animals but splenomegaly was prominent in animals treated with either agent.

Alitretinoin↗

PubMed2003

Probing the role of axial methionine in the blue copper center of azurin with unnatural amino acids.

Expressed protein ligation was used to replace the axial methionine of the blue copper protein azurin from Pseudomonas aeruginosa with unnatural amino acids. The highly conserved methionine121 residue was replaced with the isostructural amino acids norleucine (Nle) and selenomethionine (SeM). The UV-visible absorption, X- and Q-band EPR, and Cu EXAFS spectra of the variants are slightly perturbed from WT. All variants have a predominant S(Cys) to Cu(II) charge transfer band around 625 nm and narrow EPR hyperfine splittings. The Se EXAFS of the M121SeM variant is also reported. In contrast to the small spectral changes, the reduction potentials of M121SeM, M121Leu, and M121Nle are 25, 135, and 140 mV, respectively, higher than that of WT azurin. The use of unnatural amino acids allowed deconvolution of different factors affecting the reduction potentials of the blue copper center. A careful analysis of the WT azurin and its variants obtained in this work showed the large reduction potential variation was linearly correlated with the hydrophobicity of the axial ligand side chains. Therefore, hydrophobicity is the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands.

Absorptiometry, Photon↗

PubMed2003

Resonance Raman spectroscopy of cytochrome c peroxidase variants that mimic manganese peroxidase.

Cytochrome c peroxidase (C cP) variants with an engineered Mn(II) binding site, including MnC cP [C cP(MI, G41E, V45E, H181D)], MnC cP(W191F), and MnC cP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy. Analysis of the Raman bands in the 200-700 cm(-1) and 1300-1650 cm(-1) regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of C cP(MI), the recombinant yeast C cP containing additional Met-Ile residues at the N-terminus. At neutral pH the frequencies of the nu(3) mode indicate that a pure five-coordinate heme iron exists in C cP(MI) whereas a six-coordinate low-spin iron is the dominant species in the C cP variants with the engineered Mn(II) binding site. The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes. Raman spectra of the variants C cP(MI, W191F) and C cP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites. The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP. Such structural features, with a loosened distal water, may facilitate the binding of H(2)O(2) and increase the rate constant for compound I formation. This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnC cP(W191F, W51F).

Amino Acid Substitution↗