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Biomedical subjects

Y Dou

Publications and source records attributed to Y Dou.

At least 37 records · Page 2Linked to original sources
PubMed1999

Beta-amyloid peptide fragment 31-35 induces apoptosis in cultured cortical neurons.

A synthetic fragment 31-35 of beta-amyloid peptide was used in cultured cortical neurons to examine whether this smaller sequence could trigger apoptotic degeneration in vitro by using morphological, biochemical and flow-cytometric examinations. The results showed that: (i) neurons treated with fragment 31-35 of beta-amyloid peptide exhibited membrane blebbing, compaction of nuclear chromatin, nuclear shrinkage and nuclear fragmentation; (ii) a typical DNA ladder was revealed by agarose gel electrophoresis following fragment 31-35 of beta-amyloid peptide exposure; (iii) the internucleosome DNA fragmentation was also detected by flow-cytometric examination following fragment 31-35 of beta-amyloid peptide exposure; and (iv) the DNA fragmentation induced by fragment 31-35 of beta-amyloid peptide in the above two examinations could be blocked by co-treatment with aurintricarboxylic acid or actinomycin D. It is suggested that fragment 31-35 of the beta-amyloid peptide may be a shorter sequence of beta-amyloid peptide responsible for triggering an apoptotic process in cultured neurons.

Amyloid beta-Peptides↗
PubMed1999

Phosphorylation of linker histone H1 regulates gene expression in vivo by mimicking H1 removal.

Two Tetrahymena strains were created by gene replacement. One contained H1 with all phosphorylation sites mutated to alanine, preventing phosphorylation. The other had these sites changed to glutamic acid, mimicking the fully phosphorylated state. Global gene expression was not detectably changed in either strain. Instead, H1 phosphorylation activated or repressed specific genes in a manner that was remarkably similar to the effects of knocking out the gene encoding H1. These studies demonstrate a role for H1 phosphorylation in the regulation of transcription in vivo and suggest that it acts by mimicking the partial removal of H1.

Animals↗
PubMed1999

[Component in diphtheria-pertussis-tetanus-hepatitis B vaccine].

OBJECTIVE: To study immunogenecity of yeast-derived recombinant hepatitis B (YHB) component in diphtheria-pertussis-tetanus-YHB vaccine (DPTw-YHB). METHODS: Immunogenecity of tetra-valent DTPw-YHB vaccine and mono-valent recombinant YHB vaccine, and that of the tetra-valent vaccine with varied YHB component were compared. The efficiency and stability of recombinant YHB in the tetra-valent vaccine stored at 2 - 8 degrees C for 18 months were determined. RESULTS: The efficiency of recombinant YHB in the tetra-valent vaccine enhanced significantly in mice, as compared with that of mono-valent recombinant YHB vaccine, with an average mouse ED(50) of 1:2.0 - 1:3.1. There was no significant difference in efficiency of the tetra-valent vaccine with varied recombinant YHB component. Recombinant YHBin the DPTw-YHB tetra-valent vaccine still kept good stability stored at 2 - 8 degrees C for 18 months. CONCLUSION: Recombinant YHB in the tetra-valent vaccine was more immunogenic than the mono-valent YHB vaccine. No interference and inhibition of DPTw to recombinant YHB was found, indicating good compatibility between DPTw and YHB.

Animals↗
PubMed1998

Myoglobin mutants giving the largest geminate yield in CO rebinding in the nanosecond time domain.

We have measured the rebinding of carbon monoxide (CO) to some distal mutants of myoglobin (Mb) in the time range from 10(-8) to 10(-1) s by flash photolysis, in which the photodissociated CO rebinds to the heme iron without escaping to the solvent water from the protein matrix. We have found that the double mutants [His64-->Val/Val68-->Thr (H64V/V68T) and His64-->Val/Val68-->Ser (H64V/V68S)] have an extremely large geminate yield (70-80%) in water at 5 degreesC, in contrast to the 7% of the geminate yield of wild-type Mb. The CO geminate yields for these two mutants are the largest in those of Mb mutants reported so far, showing that the two mutants have a unique heme environment that favors CO geminate rebinding. Comparing the crystal structures and 1H-NMR and vibrational spectral data of H64V/V68T and H64V/V68S with those of other mutants, we discuss factors that may control the nanosecond geminate CO rebinding and CO migration in the protein matrix.

Carbon Monoxide↗
PubMed1998

[Infection of GBV-C virus among hepatitis and HCC patients].

To study the infection status of GBV-C virus among HBV, HCV, nonA-E and HCC patients, serum of clinical hepatitis patients and HCC patients before operation, and the composition in/beside HCC were collected to examine HGV RNA of the samples, using HGV RT-PCR method. Results showed that among the clinical patients with HBV, HCV, nonA-E and HCC, HGV infection rates were 9% (12/130), 10% (3/30), 17% (4/24), 0% (0/24) respectively, suggesting that HGV is a hepatitis virus with pervasive existence and has high superimposition infection rate with HBV and HCV but with little connection with HCC.

China↗
PubMed1997

Rifampicin carrying polyhydroxybutyrate microspheres as a potential chemoembolization agent.

In this study, we attempted to prepare microspheres from a microbial biodegradable polyester, i.e. polyhydroxybutyrate (PHB) as a potential chemoembolization agent. The drug loaded PHB microspheres were prepared by a solvent evaporation technique, in which methylene chloride, distilled water, and polyvinyl alcohol were utilized as the solvent, dispersion medium, and emulsifier, respectively. Microspheres were obtained within a size range of 5-100 microns by changing the initial polymer/solvent ratio, emulsifier concentration, stirring rate, and initial drug concentration. It was possible to obtain PHB with very narrow size distributions by applying gravity field-flow fractionation technique. Very high drug loadings of up to 407.6 mg rifampicin/g PHB were achieved. Drug release rates were very rapid. Almost 90% of the drug loaded was released in about 24 h. Both the size and drug content of PHB microspheres were found to be effective in controlling the drug release from these microspheres.

Antibiotics, Antitubercular↗
PubMed1997

Manifestation of Behcet's disease in the digestive tract.

OBJECTIVE: To study the manifestations of Behcet's disease (BD) in the digestive tract and its differential diagnosis from common inflammatory bowel diseases. METHODS: The symptoms and endoscopic, X-ray's or surgical characteristics of 24 cases of Behcet's disease with digestive tract manifestations were analysed. RESULTS: The lesions mainly occurred in tileocecum, and frequently led to hemorrhage that required surgery. The recurrent rate was as high as 77.7%. CONCLUSION: Importance should be attached to early diagnosis of BD of the digestive tract to prevent serious complications.

Abdominal Pain↗
PubMed1996

High resolution crystal structures of the deoxy, oxy, and aquomet forms of cobalt myoglobin.

The structures of the deoxy, oxy, and aquomet forms of native sperm whale myoglobin reconstituted with cobalt protoporphyrin IX have been determined by x-ray crystallography. As expected, cobalt myoglobin closely resembles native iron myoglobin in overall structure, especially in their respective aquomet forms. In the cobalt oxymyoglobin structure, the Nepsilon of distal histidine 64 lies within hydrogen bonding distance to both the oxygen atom directly bonded to the cobalt and the terminal oxygen atom, in agreement with previous EPR and resonance Raman studies. The metal atom in cobaltous myoglobin does show a small 0.06-A out-of-porphyrin plane displacement when moving from the oxy to deoxy state. In the case of the native iron-containing myoglobin, the oxy to deoxy transition results in a larger 0.16-A displacement of the metal farther out of the porphyrin plane, attributed to an increase in spin from S = 0 to S = 2. The small displacement in cobalt myoglobin is due to a change in coordination geometry, not spin state (S = 1/2 for both cobalt deoxy- and oxymyoglobin). The small out-of-porphyrin plane movement of cobalt which accompanies deoxygenation of myoglobin also occurs in cobalt hemoglobin and serves to explain why cooperativity, although reduced, is still preserved when iron is replaced by cobalt in human hemoglobin.

Animals↗
PubMed1996

Mechanism of hydrogen cyanide binding to myoglobin.

Cyanide binding to myoglobin is much slower than that of other ferric and ferrous ligands, suggesting rate limitation by bond formation and disruption within the distal pocket. This interpretation is supported by two key experimental observations. First, His64(E7) to Gly and Ala mutations, which open a direct channel from the solvent to the iron atom, and Phe46(CD4) to Leu, Ile, and Val mutations, which increase the mobility of the distal histidine, have little effect on the association rate constant for cyanide binding. In contrast, these mutations cause 100-1000-fold increases in the rate constant for azide binding, showing convincingly that the binding of this ligand is limited by the rate of its movement into the protein. Second, the rate constant for cyanide dissociation is unaffected by changing the size of the residue at position 64(E7) in the series Gly, Val, Leu, Ile, Phe, whereas there is a 2000-fold decrease in the rate of azide dissociation in going from Gly64 to Phe64 metmyoglobin. The major determinants of the cyanide affinity are the ease of water displacement from the ferric iron atom in metmyoglobin, the acid dissociation constant of HCN inside the protein (K*a), and steric hindrance and electrostatic interactions at the sixth coordination position. Direct hydrogen bonding to the distal histidine does not appear to play an important role in stabilizing bound cyanide. Instead, the general polarity of the distal pocket and its effect on K*a are the key factors regulating cyanide affinity under physiological conditions.

Amino Acids↗
PubMed1996

Ciliary neurotrophic factor antagonizes gentamicin-induced alterations of electric potentials in auditory pathway in guinea pigs.

AIM: To study the effects of ciliary neurotrophic factor (CNTF) on the expressions of gentamicin ototoxicity in guinea pigs. METHODS: The auditory function of pigmented guinea pigs was examined using auditory brainstem response (ABR), cochlea microphonic potential (CM), and action potential of auditory nerve (AP). RESULTS: In animals injected gentamicin (80 mg.kg-1.d-1, i.m.), ABR threshold began to elevate on d 20, and prolongations of ABR wave I, IV and the I-IV interpeak latencies were observed. The animals treated with gentamicin for 30 d displayed lower amplitudes of CM and AP (N1) than the controls. CNTF (0.44 mg.kg-1.d-1, s.c.) inhibited the gentamicin-induced elevation of ABR thresholds, the prolongation of ABR wave I, IV and the I-IV interpeak latencies, and the decreases in amplitudes of CM and AP (N1). CONCLUSION: CNTF attenuated the gentamicin-elicited auditory impairment in guinea pigs.

Action Potentials↗
PubMed1995

Alteration of axial coordination by protein engineering in myoglobin. Bisimidazole ligation in the His64-->Val/Val68-->His double mutant.

Pig and human myoglobin have been engineered to reverse the positions of the distal histidine and valine (i.e. His64(E7)-->Val and Val68(E11)-->His). Spectroscopic and ligand binding properties have been measured for human and pig H64V/V68H myoglobin, and the structure of the pig H64V/V68H double mutant has been determined to 2.07-A resolution by x-ray crystallography. The crystal structure shows that the N epsilon of His68 is located 2.3 A away from the heme iron, resulting in the formation of a hexacoordinate species. The imidazole plane of His68 is tilted relative to the heme normal; moreover it is not parallel to that of His93, in agreement with our previous proposal (Qin, J., La Mar, G. N., Dou, Y., Admiraal, S. J., and Ikeda-Saito, M. (1994) J. Biol. Chem. 269, 1083-1090). At cryogenic temperatures, the heme iron is in a low spin state, which exhibits a highly anisotropic EPR spectrum (g1 = 3.34, g2 = 2.0, and g3 < 1), quite different from that of the imidazole complex of metmyoglobin. The mean iron-nitrogen distance is 2.01 A for the low spin ferric state as determined by x-ray spectroscopy. The ferrous form of H64V/V68H myoglobin shows an optical spectrum that is similar to that of b-type cytochromes and consistent with the hexacoordinate bisimidazole hemin structure determined by the x-ray crystallography. The double mutation lowers the ferric/ferrous couple midpoint potential from +54 mV of the wild-type protein to -128 mV. Ferrous H64V/V68H myoglobin binds CO and NO to form stable complexes, but its reaction with O2 results in a rapid autooxidation to the ferric species. All of these results demonstrate that the three-dimensional positions of His64 and Val68 in the wild-type myoglobin are as important as the chemical nature of the side chains in facilitating reversible O2 binding and inhibiting autooxidation.

Animals↗
PubMed1995

Structural and functional effects of apolar mutations of the distal valine in myoglobin.

High-resolution structures of the aquomet, deoxy, and CO forms of Ala68, Ile68, Leu68, and Phe68 sperm whale myoglobins have been determined by X-ray crystallography. These 12 new structures, plus those of wild-type myoglobin, have been used to interpret the effects of mutations at position 68 and the effects of cobalt substitution on the kinetics of O2, CO, and NO binding. Molecular dynamics simulations based on crystal structures have provided information about the time-dependent behavior of photolyzed ligands for comparison with picosecond geminate recombination studies. The Val68-->Ala mutation has little effect on the structure and function of myoglobin. In Ala68 deoxymyoglobin, as in the wild-type protein, a water molecule hydrogen-bonded to the N epsilon atom of the distal histidine restricts ligand binding and appears to be more important in regulating the function of myoglobin than direct steric interactions between the ligand and the C gamma atoms of the native valine side-chain. This distal pocket water molecule is displaced by the larger side-chains at position 68 in the crystal structures of Leu68 and Ile68 deoxymyoglobins. The Leu68 side-chain can rotate about its C alpha-C beta and C beta-C gamma bonds to better accommodate bound ligands, resulting in net increases in overall association rate constants and affinities due to the absence of the distal pocket water molecule. However, the flexibility of Leu68 makes simulation of picosecond NO recombination difficult since multiple starting conformations are possible. In the case of Ile68, rotation of the substituted side-chain is restricted due to branching at the beta carbon, and as a result, the delta methyl group is located close to the iron atom in both the deoxy and liganded structures. The favorable effect of displacing the distal pocket water molecule is offset by direct steric hindrance between the bound ligand and the terminal carbon atom of the isoleucine side-chain, resulting in net decreases in affinity for all three ligands and inhibition of geminate recombination which is reproduced in the molecular dynamics simulations. In Phe68 myoglobin, the benzyl side-chain is pointed away from the ligand binding site, occupying a region in the back of the distal pocket. As in wild-type and Ala68 myoglobins, a well-defined water molecule is found hydrogen bonded to the distal histidine in Phe68 deoxymyoglobin. This water molecule, in combination with the large size of the benzyl side-chain, markedly reduces the speed and extent of ligand movement into the distal pocket. (ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

1H NMR investigation of distal mutant deoxy myoglobins. Interpretation of proximal His contact shifts in terms of a localized distal water molecule.

1H NMR spectra of a series of distal point mutants of human and sperm whale deoxy myoglobin have been recorded and their spectral parameters compared with those of wild type. The substitutions investigated include His64(E7)-->Gly, Ala, Val, Leu, Ile, and Gln and Val68(E11)-->Ala, Ile. The three resonances from the proximal His F8 imidazole ring, as well as two heme methyl signals, are identified in each of the proteins. Significant perturbations of the NMR spectra of mutant deoxy myoglobins (Mbs) occurred only upon substitution of His64(E7) by any non-polar residue, with only minor variation in parameters throughout the range of side chains. These spectral changes are attributed to the elimination of a non-coordinated ordered water molecule in the distal pocket found hydrogen bonded to His64(E7) in crystals of wild-type deoxy Mb, but abolished in the His64(E7)-->Leu mutant deoxy Mb crystal (Quillin, M. L., Arduini, R. M., Olson, J. S., and Philips, G. N., Jr. (1993) J. Mol. Biol. 234, 140-155). The observed spectral changes, increased His F8 ring spin delocalization, and decreased heme in-plane asymmetry, can be directly attributed to the weakening of the effective axial field and a decrease in the asymmetry in the rhombic ligand field resulting from removal of the water molecule. The hyperfine shift patterns for the mutants His64(E7)-->Gln and Val68(E11)-->Ile deoxy Mbs are minimally perturbed from that of wild type and are interpreted to reflect a conserved distal water-binding site. In the point mutant Val68(E11)-->Ala, the decreased covalency to the axial His F8 is interpreted as reflecting a conserved distal water molecule that can interact more strongly with the iron due to the reduced steric bulk of the E11 side chain. The differential 1H NMR spectral parameters for the His F8 resonances in the two subunits of T state deoxy Hb A are shown to be similarly consistent with the known occupation of the distal water binding site in the alpha-, but not beta-subunit.

Animals↗
PubMed1994

Stability of myoglobin: a model for the folding of heme proteins.

Factors governing the stability of sperm whale, pig, and human metmyoglobin were examined by (1) measuring guanidinium chloride induced unfolding of apoglobins containing 22 replacements at positions 29(B10), 43(CD1), 64(E7), 68(E11), and 107(G8), (2) determining the rates of hemin loss from the recombinant holoproteins, and (3) estimating constitutive expression levels of the corresponding genes in Escherichia coli TB-1 cells. The denaturant titrations were analyzed in terms of a two-step unfolding reaction, N(native apoprotein)-->I(intermediate)-->U(unfolded), in which the intermediate is visualized by an increase in tryptophan fluorescence emission. Two key conclusions were reached. First, high rates of hemin loss are not necessarily correlated with unstable globin structures and vice versa. In general, both rates of hemin loss and the equilibrium constants for apoprotein unfolding must be determined in order to understand the overall stability of heme proteins and to predict the efficiency of their expression. Second, polar residues in the distal pocket cause marked decreases in the overall stability of apomyoglobin. Removal of hemin from V68N and L29N sperm whale myoglobins produces the molten globular I state at pH 7, 25 degrees C, without addition of denaturant. In contrast, the H64L and H64F mutations produce apoproteins which are 10-30 times more stable than wild-type apoglobin. The latter results show that protein stability is sacrificed in order to have the distal histidine (H64) present to increase O2 affinity and inhibit autooxidation.

Animals↗
PubMed1994

Electron-nuclear coupling to the proximal histidine in oxy cobalt-substituted distal histidine mutants of human myoglobin.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to investigate electron-nuclear coupling to the N epsilon of the proximal histidine (F8, His93) imidazole in oxyCo(II)-substituted distal histidine (E7, His64) mutants (His-->Leu, His-->Val, His-->Gly, His-->Gln) and recombinant wild-type human myoglobins (Mbs). Nuclear hyperfine and nuclear quadrupole coupling constants decrease in the order: H64L > H64V > or = H64G approximately H64Q > wild-type. The differences in couplings found for the four mutant proteins are correlated with the differences in polarity of the E7 side chain. On the basis of the relative orientation of the nuclear quadrupole and g tensors, obtained by computer simulation of ESEEM spectra, the Co-O-O bond angle of H64G and H64Q appears to be similar to that of oxyCo sperm whale Mb (and possibly wild-type human Mb) at room temperature [Hori et al. (1982) J. Biol. Chem. 257, 3636], while that in H64V and H64L is more obtuse. ESEEM measurements in D2O demonstrate the presence of a hydrogen bond between the distal histidine and bound O2 in the wild-type protein, as was found in oxyCo sperm whale and horse Mbs [Lee et al. (1992) Biochemistry 31, 7274]. This hydrogen bond leads to a reduction in the N epsilon coupling in the wild-type protein as compared to that in the E7 mutants. No hyperfine-coupled deuterons were found in any of the mutants, and therefore, the proposed hydrogen bond between bound O2 and the distal glutamine in H64Q [Ikeda-Saito et al. (1991) J. Biol. Chem. 266, 23641] could not be substantiated.

Chemical Phenomena↗
PubMed1994

Structural factors governing azide and cyanide binding to mammalian metmyoglobins.

The structural factors governing azide and cyanide binding have been examined by measuring the effects of 46 mutations at key topological positions in the distal pocket in sperm whale, pig, and human myoglobin. Replacement of His64 (E7) with smaller amino acids results in dramatic increases in the association rate constant for azide binding primarily due to relief of steric hindrance imposed by the imidazole side chain. Gln64 and His64 (native) metmyoglobins have abnormally low rate constants for azide dissociation (0.1-0.3 s-1) due to direct hydrogen bonding between the N epsilon atoms of these residues and the bound ligand. Mutations at positions 67(E10) and 68(E11) produce large but complex changes in the azide binding parameters as a result of both steric and electrostatic effects, which alter water coordination, influence the rate of anion movement into the distal pocket, and affect the stability of the Fe-N3 bond. Replacement of Phe46 with Leu or Val and substitution of Arg(Lys)45 with Glu and Ser cause disorder in the position of the distal histidine side chain and result in 4-700-fold increases in both k'N3 and kN3 but produce little change in overall azide affinity. All of these results suggest strongly that azide enters the distal pocket of native myoglobin through a polar channel that is regulated by a His64 "gate." In contrast to azide binding, the rate constant for cyanide association decreases 4-300-fold when the distal histidine is replaced with apolar residues. His64, Gln64, and distal pocket water molecules appear to facilitate deprotonation of HCN, which is the major kinetic barrier to cyanide binding at neutral pH.

Animals↗
PubMed1994

1H NMR study of the solution molecular and electronic structure of engineered distal myoglobin His64(E7) Val/Val68(E11) His double mutant. Coordination of His64(E11) at the sixth position in both low-spin and high-spin states.

A genetically engineered human myoglobin (Mb) in which the distal His, His64(E7), and the distal Val, Val68(E11), are replaced by Val and His, respectively, has been expressed in Escherichia coli, for the purpose of assessing the potential role of a E11 residue in providing a hydrogen bond donor to the coordinated ligand. Molecular modeling indicates that such an interaction is possible. The 1H NMR spectrum of the ferric form of the double mutant Mb exhibits large hyperfine shifts and strong paramagnetic relaxation for which the temperature dependence of the hyperfine shifts reveals a thermal equilibrium between a low-spin and high-spin state (70, 30% at 25 degrees C, respectively). Standard sequence specific two-dimensional (2D) NMR assignments of the E and F helical backbones allow the identification of the peptide protons for the proximal His93(F8) and substituted distal His68(E11). Steady-state nuclear Overhauser effect from these peptide protons locate strongly hyperfine shifted His93(F8) and His68(E11) side chain protons which dictate that both the imidazole rings are coordinated to the iron. 2D bond correlation and one-dimensional and 2D dipolar correlation experiments locate and assign the resonances for the heme. The pattern of the heme contact shifts in both the low-spin and high-spin state, together with the nature of the temperature dependence of the His93(F8) and His68(E11) resonances, establish that the two His are ligated in the high-spin as well as low-spin forms. The pattern of heme methyl hyperfine shifts in the low-spin state, and the smaller hyperfine shifts for His68(E11) as compared to His93(F8) in the high-spin state, indicate that the axial bond to the distal His68(E11) is weakened or strained as compared with that for the proximal His93(F8) in both spin states. This weak ligation originates from a tilted iron-His68 bond, the only conformation in which His68 can place its imidazole group sufficiently close to bind to the heme iron in the conventional Mb folding. Not only do these results support the belief that distal His is indispensable for the control of the ligand binding in Mb and hemoglobin, but also reveal the significance of the evolution that the stereochemical disposition of both His64 and Val68 are unique and non-exchangeable for interacting with the bound ligand.

Amino Acid Sequence↗