Biomedical subjects

Y Dou

Publications and source records attributed to Y Dou.

At least 19 recordsLinked to original sources

PubMed2003

Pre-neutralization of C5a-mediated effects by the monoclonal antibody 137-26 reacting with the C5a moiety of native C5 without preventing C5 cleavage.

Complement C5a is aetiologically linked to inflammatory tissue damage in conditions like septicaemia, immune complex diseases and ischaemia-reperfusion injury. We here describe a monoclonal antibody (mAb), 137-26, that binds to the C5a moiety of human C5 and neutralizes the effects of C5a without interfering with C5 cleavage and the subsequent formation of lytic C5b-9 complex. Mouse anti-human C5 mAbs were generated and the reactivity with C5 and C5a was detected by ELISA and surface plasmon resonance. The inhibition of C5a binding to C5a receptor was studied using a radioligand binding assay. The effects of the antibody on C5a functions were examined using isolated neutrophils and a novel human whole blood model of inflammation. Haemolytic assays were used to study the effect on complement-mediated lysis. mAb 137-26 reacted with both solid- and solution-phase C5 and C5a in a dose-dependent manner with high affinity. The antibody competed C5a binding to C5a receptor and inhibited C5a-mediated chemotaxis of neutrophils. Furthermore, the antibody effectively abrogated complement-dependent E. coli-induced CD11b up-regulation and oxidative burst in neutrophils of human whole blood. mAb 137-26 was more potent than a C5a receptor antagonist and a previously described anti-C5a antibody. mAb 137-26 did not inhibit complement-mediated lysis, nor did it activate complement itself. Together, mAb 137-26 binds both the C5a moiety of native C5 and free C5a, thereby effectively neutralizing the biological effects of C5a. The antibody may have therapeutic potential in inflammatory diseases where C5a inhibition combined with an operative lytic pathway of C5b-9 is particularly desired.

Animals↗

PubMed2002

Morphometric and biomechanical remodelling in the intestine after small bowel resection in the rat.

The short-bowel syndrome is a clinical condition caused by intestinal resection. As intestinal adaptation occurs after resection, it can be used as a model for studying morphometric and biomechanical remodelling in the small intestine and to get a better understanding of the pathophysiology of the short-bowel syndrome. The resected rats had a 67% resection of jejunum and ileum. Control animals underwent no operation (nonoperated controls) or an ileal transection with subsequent end-to-end anastomosis (sham-resected controls). The animals were followed for up to 4 weeks after the operation. Changes in biomechanical properties were studied in terms of residual strain (the internal strain remaining when all external loads are removed), opening angle and stress--strain relations referenced to the zero-stress state (the cut-open state where external and internal stresses are released). The resected animals gained less weight than the controls. The intestinal length and diameter increased more in the resected groups than the control groups (P < 0.05), resulting in a larger absorptive surface. Resection induced profound gross morphometric changes and histological alterations characterized by proliferative increases in the tissue layers. The opening angle, along with residual strain at the mucosal and serosal surface, increased in the remnant small intestine (P < 0.05). All changes increased as function of postoperative time and were most prominent in the remnant ileum. However, the stress-strain relationship remained unchanged. In conclusion, this study demonstrated that resection of the majority of the small bowel results in significant remodelling in structural and residual strain properties in the remnant small intestine. The remodelling seems to be guided by the need for a greater absorptive surface area rather than for a change in the stress-strain properties.

Adaptation, Physiological↗

PubMed2002

Testosterone and estradiol modulate TNF-alpha-induced expression of adhesion molecules in endothelial cells.

Cytokine-activated endothelial expression of adhesion molecules plays an important role in immune responses. In the present study, we investigated the influences of testosterone and 17 beta-estradiol on tumor necrosis factor-alpha (TNF-alpha)-induced expression of adhesion molecules in human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with TNF-alpha, testosterone or 17 beta-estradiol separately, or in a combination of TNF-alpha plus testosterone or 17 beta-estradiol. The expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was evaluated at 3, 6, 12 and 24 h following exposure by flow cytometric analysis. The results showed that although testosterone or 17 beta-estradiol did not affect the expression of these adhesion molecules in unstimulated HUVEC, both of them transiently increased the expression of E-selectin and VCAM-1 in TNF-alpha stimulated HUVEC. Neither testosterone nor 17 beta-estradiol affected the expression of ICAM-1 induced by TNF-alpha. It is concluded that both testosterone and 17 beta-estradiol increase TNF-alpha-induced expression of E-selectin and VCAM-1 in endothelial cells and these facts might indicate a mechanism by which gonadal hormones can indirectly enhance immune responses.

Cells, Cultured↗

PubMed2001

Effect of re-feeding after starvation on biomechanical properties in rat small intestine.

Luminal nutrients are essential for maintaining the structural and functional integrity of the gut. Starvation induces pronounced structural and biomechanical remodelling in the rat small intestine. The present work was done to study the recovery process after resumption of food intake. Twenty-five Wistar rats were allocated to five groups. Four groups fasted for 7 days but had free access to water. One of these groups served as fasted controls and was killed at the end of the fast. The other three groups were re-fed for 2, 4 and 7 days before they were euthanised. The fifth group had free access to food during the whole study (fed controls). The intestinal no-load state, zero-stress state and the stress-strain relationship during distension were studied. The intestinal segments were cut transversely into a series of short ring-shaped segments to obtain the no-load state. Each ring was cut in the radial direction to obtain the zero-stress state. The rats regained the lost body weight (22%) by the 7th day of re-feeding. The lost duodenal mass (40%) and jejunal mass (25%) were regained by the 2nd day whereas the lost mass from ileum (18%) was regained by the 4th day. The fasting-induced morphometric changes were normalised by re-feeding on the 2nd day in the duodenum and jejunum, and on the 4th day in the ileum. The longitudinal stress-strain curves shifted to the right after fasting and shifted back within two days following re-feeding (P<0.05). The circumferential stress-strain curves in the fasted or re-fed rats changed in a similar though less pronounced way. Normal values were reached within 4-7 days for the circumferential direction. In conclusion, fasting-induced biomechanical and structural remodelling were normalised by re-feeding in a time- and location-dependent way.

Animals↗

PubMed2001

[An experimental study on the effects of early postburn nutrition on calcitonin gene--related peptide].

OBJECTIVE: To explore the resuscitation effects of different nutritional routes during early postburn stage on intestinal tract, and the relationship between calcitonin gene -- related peptide (CGRP) and resuscitation of intestinal tract. METHODS: Sixty Wistar rats inflicted by 30% TBSA III degree flame burn were randomly divided into burn with enteral feeding group (EF, 30 rats) and burn with early parenteral nutrition group (EPN group, 30 rats). Six normal rats without burn injury served as control group (C). The changes in general condition, intestinal mucosal blood flow, plasma concentration of CGRP immunologic competence substance and intestinal CGRP positive staining substance in the rats were observed at 6, 12, 24, 48 and 72 postburn hours (PBHs). RESULTS: The rat intestinal mucosal blood flow decreased postburn, especially in EPN group (P < 0.05). The postburn plasma CGRP increased and the plasma CGRP level in EF group was obviously lower than that in EPN group at 72 PBH (P < 0.01). The CGRP positive staining cells of intestinal tract decreased markedly postburn (P < 0.05) in both burn groups, and those in EPN were significantly lower abundant than those in EF group at 24 and 48 PBHs (P < 0.05). Furthermore, the decrease in intestinal mucosal blood flow was positively related to the decrease in intestinal intramuscular nerve plexus CGRP (r = 0.72, P < 0.05). CONCLUSION: Enteral nutrition exerted better effects on early postburn intestinal resuscitation than parenteral nutrition. The decrease in intestinal blood supply might be mainly due to the excessive increase in plasma CGRP immune active substance and the decrease of postburn intestinal tract CGRP immunologic competence substance. Early enteral feeding might play resuscitative role by modulating CGRP.

Animals↗

PubMed2001

Studies on the development of DNA vaccine against Cysticercus cellulosae infection and its efficacy.

DNA vaccine against Cysticercus cellulosae infection was developed and its efficacy was tested. A pair of primers specific to antigen B gene of C. cellulosae was designed which amplified the gene successfully with RT-PCR. The gene was ligated to PV93 vector, and the recombinant of antigen B gene and PV93 was transformed to JM83 cells. The transformed JM83 cells were cultured in a large scale and the plasmid purified. Based on the recombinant plasmid. a DNA vaccine was developed and used to vaccinate two groups of experimental pigs. In each group, there was a routine vaccine, an enhanced vaccine and a control group. Groups 1 and 2 were challenged at 4 months and at 14 days post vaccination respectively with eggs of Taenia solium. The antibody response was also tested with ELISA. The results suggested that all animals vaccinated AgB gene DNA vaccine, no matter by routine or enhanced vaccine, their antibodies reached maximum peak 23 days post vaccination and decreased gradually. When the animals were challenged 4 months after vaccination, they had strong immunity and the parasites decrease rates were 91.2% and 93.1% respectively. When pigs vaccinated with AgB gene DNA vaccine were challenged 14 days post vaccination with 18,000 eggs/pig. The animals showed strong immunity and the parasite decrease rates were 99.5% and 84.9% respectively. However at that time, the antibodies did not reach the peak. While in the control group, the number of C. cellulosae was as many as 2,500. It was concluded that the pigs vaccinated with DNA vaccine had strong immunity against infection of eggs of T. solium.

Animals↗

PubMed2001

[Calcium dependent synaptic plasticity].

Recent work shows that the intracellular free Ca2+ concentrations ([Ca2+]i) of the presynaptic and postsynaptic neurons play crucial signaling roles in short- and long-term synaptic plasticity. Residual [Ca2+]i followed conditioning stimulation may cause short-term synaptic enhancement. Presynaptic [Ca2+]i could influence the replacing of presynaptic depressed vesicles, as well as encode the precise relative timing of presynaptic input and postsynaptic activity and generate long-term synaptic modification of opposite polarity(LTP or LTD).

Animals↗

PubMed2000

Waterproofing the heme pocket. Role of proximal amino acid side chains in preventing hemin loss from myoglobin.

The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group. Globin side chains at the Leu(89)(F4), His(97)(FG3), Ile(99)(FG5), and Leu(104)(G5) positions on the proximal side of the heme pocket strongly influence heme affinity. The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography. Leu(89) and His(97) are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring. Ile(99) and Leu(104) are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group. The apolar amino acids Leu(89), Ile(99), and Leu(104) "waterproof" the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment. Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss. Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His(93)-Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.

Amino Acid Substitution↗

PubMed2000

Nitric-oxide dioxygenase activity and function of flavohemoglobins. sensitivity to nitric oxide and carbon monoxide inhibition.

Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from Saccharomyces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar spectra, O(2), NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics. Turnover numbers (V(max)) for S. cerevisiae, A. eutrophus, and E. coli flavoHbs are 112, 290, and 365 NO heme(-1) s(-1), respectively, at 37 degrees C with 200 microm O(2). The K(M) values for NO are low and range from 0.1 to 0.25 microm. V(max)/K(M)(NO) ratios of 900-2900 microm(-1) s(-1) indicate an extremely efficient dioxygenation mechanism. Approximate K(M) values for O(2) range from 60 to 90 microm. NO inhibits the dioxygenases at NO:O(2) ratios of > or =1:100 and makes true K(M)(O(2)) values difficult to determine. High and roughly equal second order rate constants for O(2) and NO association with the reduced flavoHbs (17-50 microm(-1) s(-1)) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O(2) (K(I)(CO) = approximately 1 microm). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O(2) from inhibitory NO and CO.

Alcaligenes↗

PubMed2000

Steady-state and transient kinetics of Escherichia coli nitric-oxide dioxygenase (flavohemoglobin). The B10 tyrosine hydroxyl is essential for dioxygen binding and catalysis.

Escherichia coli expresses an inducible flavohemoglobin possessing robust NO dioxygenase activity. At 37 degrees C, the enzyme shows a maximal turnover number (V(max)) of 670 s(-1) and K(m) values for NADH, NO, and O(2) equal to 4.8, 0.28, and approximately 100 microM, respectively. Individual reduction, ligand binding, and NO dioxygenation reactions were examined at 20 degrees C, where V(max) is approximately 94 s(-1). Reduction by NADH occurs in two steps. NADH reduces bound FAD with a rate constant of approximately 15 microM(-1) s(-1), and heme iron is reduced by FADH(2) with a rate constant of 150 s(-1). Dioxygen binds tightly to reduced flavohemoglobin, with association and dissociation rate constants equal to 38 microM(-1) s(-1) and 0.44 s(-1), respectively, and the oxygenated flavohemoglobin dioxygenates NO to form nitrate. NO also binds reversibly to reduced flavohemoglobin in competition with O(2), dissociates slowly, and inhibits NO dioxygenase activity at [NO]/[O(2)] ratios of 1:100. Replacement of the heme pocket B10 tyrosine with phenylalanine increases the O(2) dissociation rate constant approximately 80-fold and reduces NO dioxygenase activity approximately 30-fold, demonstrating the importance of the tyrosine hydroxyl for O(2) affinity and NO scavenging activity. At 37 degrees C, V(max)/K(m)(NO) is 2,400 microM(-1) s(-1), demonstrating that the enzyme is extremely efficient at converting toxic NO into nitrate under physiological conditions.

Amino Acids↗

PubMed2000

EPR studies on the photoinduced intermediates of NO complexes in recombinant ferric-Mb trapped at low temperatures.

The nitrosyl complex of ferric myoglobin is EPR-silent. Upon photolysis at low temperatures, the photoinduced intermediates trapped in the distal heme cavity exhibit new EPR spectra due to the interaction between the photodissociated NO (S=1/2) and the ferric high spin heme (S=5/2). In order to elucidate the effect of distal E7 (His64) and E11 (Val68) mutations upon the electronic structure of the metal center, its immediate environment, and its interaction with the photodissociated NO, EPR spectra of the photoproducts of the NO complexes of recombinant ferric Mb mutants were measured at 5 K. EPR spectra of the photoproducts were closely related to the size and/or the polarity of the distal pocket residues. The distal pocket of the E7 mutants seemed to be sterically crowded, even decreasing the side chain volume or changing its hydrophobicity by replacing amino acid at position 64. We have found that the mobility of the photodissociated NO molecule in the distal heme pocket was strongly governed by the nature of the amino acid residue at E11 position.

Animals↗

PubMed2000

Carbachol blocks beta-amyloid fragment 31-35-induced apoptosis in cultured cortical neurons.

We reported previously that many neurodegenerative changes characteristic of apoptosis could be induced by a short fragment of beta-amyloid protein, A(beta31-35), in cultured newborn mice cortical neurons, and that these changes were accompanied with alterations in expression of some genes. This study was designed to examine whether the apoptotic processes and related gene modulations in this model could be affected by coadministration of carbachol by electrophoretic analysis for DNA ladder formation and by RT-PCR assays for genomic modulation. The results showed that (1) simultaneous incubation with carbachol dose- and time-dependently blocked the specific DNA ladder formation induced by exposure to A(beta31-35) and (2) the A(beta31-35)-induced downregulation of bcl-2 and upregulations of bax, p53, and c-fos genes were reversed or ameliorated by the coadministration of carbachol. It is proposed that A(beta31-35)-induced apoptosis can be prevented by carbachol through mechanisms that modulate the expression of related genes.

Amyloid beta-Peptides↗

PubMed2000

Phosphorylation of linker histone H1 regulates gene expression in vivo by creating a charge patch.

In Tetrahymena, histone H1 phosphorylation can regulate transcription and mimics loss of H1 from chromatin. We investigated the mechanism by which H1 phosphorylation affects transcription. Tetrahymena strains were created containing mutations in H1 that mimicked the charge of the phosphorylated region without mimicking the structure or increased hydrophilicity of the phosphorylated residues. Whenever the charge resembled that of the phosphorylated state, the induced expression of the CyP1 gene was greatly inhibited. Whenever the charge was similar to that of the dephosphorylated state, the CyP1 gene was induced normally. These results argue strongly that phosphorylation of H1 acts by changing the overall charge of a small domain, not by phosphate recognition or by creating a site-specific charge.

Amino Acid Sequence↗

PubMed2000

[Clinical analysis of 20 cases of adenoidal hypertrophy in adult].

OBJECTIVE: To discuss the diagnosis and properly treatment to the adult adenoidal hypertrophy. METHOD: To do nasal endoscopic and pathologic exam to all the 20 symptomatic patients before conservative or operative treatment. RESULT: The results of exam under endoscope were that 8 patients were hypertrophy I, 8 hypertrophy II, 4 hypertrophy III. Pathologic exam: 7 acute and chronic inflammation, 13 chronic inflammation with hyperplasia. TREATMENT: 10 of them received conservative treatment all have negative result. 6 received traditional operation, 2 of them reoccurred within 1 year. 4 received adenoidectomy and microwave treatment under nasal endoscope, all have good result in the next 1 year. CONCLUSION: Some adults have different sizes of adenoidal hypertrophy. This hypertrophy is different with children's under the pathologic microscope. Operative treatment especially the nasal endoscopic adenoidectomy and microwave treatment is a safe and effective method.

Adenoidectomy↗

PubMed1999

Assignment of the heme axial ligand(s) for the ferric myoglobin (H93G) and heme oxygenase (H25A) cavity mutants as oxygen donors using magnetic circular dichroism.

UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4 degreesC. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known five-coordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (FeIIIPPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of FeIIIPPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.

Alanine↗

PubMed1999

Identification and mutation of phosphorylation sites in a linker histone. Phosphorylation of macronuclear H1 is not essential for viability in tetrahymena.

Linker histone phosphorylation has been suggested to play roles in both chromosome condensation and transcriptional regulation. In the ciliated protozoan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase. Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that requirements for phosphorylation of macronuclear H1 may be limited to transcriptional regulation. Here we report the major sites of phosphorylation of macronuclear H1 in Tetrahymena thermophila. Five phosphorylation sites, present in a single cluster, were identified by sequencing 32P-labeled peptides isolated from tryptic peptide maps. Phosphothreonine was detected within two TPVK motifs and one TPTK motif that resemble established p34(cdc2) kinase consensus sequences. Phosphoserine was detected at two non-proline-directed sites that do not resemble known kinase consensus sequences. Phosphorylation at the two noncanonical sites appears to be hierarchical because it was observed only when a nearby p34(cdc2) site was also phosphorylated. Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites were viable even though macronuclear H1 phosphorylation was abolished. These data suggest that the five sites identified comprise the entire collection of sites utilized by Tetrahymena and demonstrate that phosphorylation of macronuclear H1, like the protein itself, is not essential for viability in Tetrahymena.

Amino Acid Sequence↗

PubMed1999

Heme protein dynamics revealed by geminate nitric oxide recombination in mutants of iron and cobalt myoglobin.

Nitric oxide myoglobin (MbNO) at 300 K was photodissociated with 405 nm pulses. The NO recombination in several mutants of iron and cobalt myoglobins was investigated at a time resolution of ca. 70 fs. The geminate recombination of NO was nonexponential on sub-nanosecond time scales. For both metals, the change of the detailed structure of the heme pocket (position 68 mutations) caused significant changes in the rates of recombination; however, the metal substitution influenced the recombination much less than did amino acid substitution. The results indicate a primary role of the heme pocket structure in the dynamics, and they suggest that proximal protein relaxation is not the limiting factor in the geminate recombination process. Recombination in cobalt derivatives is somewhat more efficient on the sub-nanosecond time scales than in corresponding iron myoglobins, consistent with other results that show a greater intrinsic reactivity toward the NO of cobalt compared with the iron heme. A comparison of results using Soret band excitation with previous Q-state excitation studies demonstrates that the ligand dissociates with a similar kinetic energy in both cases, suggesting fast intramolecular energy redistribution before dissociation.

Amino Acid Substitution↗

PubMed1999

A cyanobacterial hemoglobin with unusual ligand binding kinetics and stability properties.

The glbN gene of the cyanobacterium Nostoc commune UTEX 584 encodes a hemoprotein, named cyanoglobin, that has high oxygen affinity. The basis for the high oxygen affinity of cyanoglobin was investigated through kinetic studies that utilized stopped-flow spectrophotometry and flash photolysis. Association and dissociation rate constants were measured at 20 degrees C for oxygen, carbon monoxide, nitric oxide, and methyl and ethyl isocyanides. The association rate constants for the binding of these five ligands to cyanoglobin are the highest reported for any naturally occurring hemoglobin, suggesting an unhindered and apolar ligand binding pocket. Cyanoglobin also shows high rates of autoxidation and hemin loss, indicating that the prosthetic group is readily accessible to solvent. The ligand binding behavior of cyanoglobin was more similar to that of leghemoglobin a than to that of sperm whale myoglobin. Collectively, the data support the model of cyanoglobin function described by Hill et al. [(1996) J. Bacteriol. 178, 6587-6598], in which cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal.

Bacterial Proteins↗