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Y Q He

Publications and source records attributed to Y Q He.

31 records · Page 2Linked to original sources
PubMed1999

Use of chimeric enzymes and site-directed mutagenesis for identification of three key residues responsible for differences in steroid hydroxylation between canine cytochromes P-450 3A12 and 3A26.

Canine cytochromes P-450 3A12 and 3A26 differ by 22 out of 503 amino acid residues. Chimeric constructs and site-directed mutants were used to identify the residues responsible for the much higher rates of steroid hydroxylation by 3A12. Six initial 3A12/3A26 hybrids were generated using convenient restriction sites, and site-directed mutagenesis was used to restore full 3A12 activity to two of the hybrids. One pair of 3A12/3A26 chimeras indicated that the first four residue differences between 3A12 and 3A26 were at least partially responsible for the differences in progesterone hydroxylation. Conversion in one of the hybrids of the Ile-187 residue found in 3A26 to the Thr in 3A12 conferred 3A12 levels of progesterone 6beta-hydroxylase activity. Analysis of another chimera identified key residues within an internal PstI fragment (codons 331-459) containing six amino acid residue differences. Subsequent site-directed mutagenesis of 3A26 residues Ser-368 and Val-369 to Pro and Ile, respectively, restored the rate of formation of 6beta-hydroxyprogesterone by the hybrid to that of 3A12. The simultaneous conversion of 3A26 residues 187, 368, and 369 to those of 3A12 conferred greater than a third of the progesterone 6beta-hydroxylase activity and all of the testosterone and androstenedione 6beta-hydroxylase activity of 3A12. Addition of the carboxyl terminal 44 3A12 residues to the 3A26 triple mutant doubled progesterone 6beta-hydroxylase activity. This is the first study to use catalytically distinct cytochromes P-450 3A from the same species in the elucidation of structure-function relationships.

Androstenedione↗
PubMed1999

An evaluation of a new gel-coupled ultrasound device for the quantitative assessment of bone.

Quantitative ultrasound (QUS) is now being accepted as a tool in the assessment of bone status. Most QUS devices measure broadband ultrasound attenuation (BUA) and speed of sound (SOS). A newly introduced device, the Acoustic Osteo-Screener (AOS-100), measures both SOS and an attenuation related parameter called the transmission index (TI) and provides a derived parameter called osteo sono-assessment index (OSI). The purpose of this study was to examine the reproducibility of this new device, compare the results with the UBA 575+ and evaluate the effect of using two platforms to compensate for different foot sizes on the measured values. 83 subjects aged 21-89 years, who gave informed consent, were recruited. Subjects were assigned to three different groups determined by age and health status. The short-term measurement precision (expressed as root mean square coefficient of variation) of SOS (0.13-0.16%) and TI (1.18-1.96%) was similar to and better than that obtained for the UBA 575+. Significant negative correlations were found between the differences in SOS measured with the two platforms (adaptors to adjust the position of the foot relative to the foot plate) and foot length (p < 0.0001). This implies that small feet are more influenced by the differences of measured location. All QUS parameters measured with the small platform were found to be significantly smaller than those with the large platform (p < 0.0001). The mean percentage differences were 0.6% in SOS, 2.8% in TI and 4.3% in OSI with the large foot platform giving larger value results. Proper use of the platforms resulted in more reproducible SOS and TI. This study demonstrated that the newly developed AOS-100 parameters TI and OSI were highly reproducible. This study also demonstrated that the use of an inappropriate platform can cause discrepancies in QUS readings and poor reproducibility.

Adult↗
PubMed1998

Structural determinants of progesterone hydroxylation by cytochrome P450 2B5: the role of nonsubstrate recognition site residues.

The highly related rabbit cytochromes P450 2B4 and 2B5 differ in only 12 amino acid positions, but only 2B5 has activity toward progesterone. Previously, simultaneous site-directed mutagenesis of four key substrate recognition site (SRS) residues (114, 294, 363, and 367) was shown to result in interconversion of the androstenedione hydroxylase specificities of cytochrome P450 2B4 and 2B5. However, the progesterone metabolite profiles of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue were not identical to that of P450 2B5. Therefore, single mutants of P450 2B5 at the remaining seven positions were constructed, expressed in Escherichia coli, and studied with progesterone as the substrate. The single mutants at positions 120 and 221, which are outside any known SRS, exhibited a significant alteration in progesterone hydroxylation. Based on these results, Ile-114, Arg-120, Ser-221, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 were replaced simultaneously with Phe, His, Pro, Thr, Val, and Ala, respectively, from 2B5. This yielded a mutant with a very similar progesterone metabolite profile to that of 2B5, although the total activity was lower. An additional substitution at residue 370 produced a multiple mutant P450 2B4 I114F-R120H-S221P-S294T-I363V-V367A- T370M with very similar or identical substrate specificity, regio- and stereospecificity and kinetic properties to that of P450 2B5 wild type.

Animals↗
PubMed1998

Osteoarthrosis of the temporomandibular joint induced by intra-articular injection of lactate dehydrogenase: an experimental study in adult rabbits.

OBJECTIVE: The purpose of this study was to establish an animal model of osteoarthrosis in the temporomandibular joint (TMJOA) by injecting lactate dehydrogenase (LDH) into the maxillary compartments of TMJs in adult rabbits. METHODS: Adult New Zealand white rabbits were randomly divided into four groups (A through D) of five animals each. The A and B groups were used as a control. The maxillary compartments of the C and D group (TMJs) were injected with 0.1 mL of LDH in two different concentrations. The TMJ samples were analyzed histologically and with a scanning electron microscope four different times after injection. RESULTS: The animals that had been injected with LDH at two different concentrations showed similar results. The TMJs had changes typical of OA in early to moderate stages. No pathologic changes could be found in the TMJs of the control groups. CONCLUSIONS: The results suggest that LDH may be of use in establishing an animal model of TMJOA.

Animals↗
PubMed1997

Identification of three key residues in substrate recognition site 5 of human cytochrome P450 3A4 by cassette and site-directed mutagenesis.

Cassette mutagenesis and site-directed mutagenesis were used to investigate the importance of individual amino acid residues at positions 364-377 of cytochrome P450 3A4 in determining steroid hydroxylation or stimulation by alpha-naphthoflavone. The mutants were expressed in an Escherichia coli system, and solubilized membranes were prepared. All mutants except R365G and R365K exhibited anti-3A immunoreactivity on Western blotting, although R372S and R375K were not detected as the Fe2+-CO complex. Replacement of Arg-372 by Lys yielded a typical P450 spectrum. The results indicate that the highly conserved Arg residues at positions 365 and 375 may play a role in stabilizing the tertiary structure or in heme binding. Catalytic activities of 12 mutants were examined using progesterone and testosterone as substrates, and residues 369, 370, and 373 were found to play an important role in determining substrate specificity. Although the three mutants hydroxylated progesterone and testosterone primarily at the 6beta-position like the wild-type, replacement of Ile-369 by Val suppressed progesterone 16alpha-hydroxylase activity, whereas substitution of Ala-370 with Val enhanced progesterone 16alpha-hydroxylation. Interestingly, substitution of Leu-373 with His resulted in production of a new metabolite from both steroids. Moreover, the mutants at positions 369 and 373 were more and less responsive, respectively, than the wild-type to alpha-naphthoflavone stimulation. Alterations in activities or expression of several mutants were interpreted using a three-dimensional model of P450 3A4. The results suggest that analogy with mammalian family 2 and bacterial cytochromes P450 can be used to predict P450 3A residues that contribute to regiospecific steroid hydroxylation.

Amino Acid Sequence↗
PubMed1997

[Determination of sinomenine HCl in serum and urine by HPLC and its pharmacokinetics in normal volunteers].

A RP-HPLC method was developed to determine the concentrations of sinomenine HCl in serum and urine and its pharmacokinetics was studied in healthy volunteers. C18H37 column was eluted with the mobile phase of acetonitrile--0.01 mol.L-1 sodium phosphate monobasic--N, N, N', N'-tetramethylenediamine (46:54:0.22 v/v, pH 6.9) and the ultraviolet absorbance was monitored at 263 nm. Triazolan was used as internal standard. The calibration curves were linear in the range of 6-480 ng.ml-1 in serum and 0.06-3 micrograms.ml-1 in urine, with mean recoveries of 75.46% and 91.38% respectively. The lowest detectable limits were 4 ng.ml-1 in serum and 40 ng.ml-1 in urine and the RSD for the intra-day and inter-day were less than 5%. A single oral dose of 80 mg sinomenine HCl tablet was given to 8 healthy male volunteers. The concentrations of sinomenine HCl in serum and urine were determined. The serum concentration--time curve was found to fit a two-compartment open model with first order elimination. The pharmacokinetic parameters were: T1/2 alpha 0.791 +/- 0.491 h, T1/2 beta 9.397 +/- 2.425 h, Tmax 1.040 +/- 0.274 h, Cmax 246.604 +/- 71.165 ng.ml-1, AUC 2651.158 +/- 1039.050 ng.h.ml-1, CL 0.033 +/- 0.010 ng.ml-1.

Adult↗
PubMed1996

Interconversion of the androstenedione hydroxylase specificities of cytochromes P450 2B4 and 2B5 upon simultaneous site-directed mutagenesis of four key substrate recognition residues.

Based on recent studies of single reciprocal mutants of cytochrome P450 2B4 and the highly related P450 2B5 at positions 114, 294, 363, and 367 [G. D. Szklarz, Y. Q. He, K. M. Kedzie, J. R. Halpert, and V. L. Burnett (1996) Arch. Biochem. Biophys. 327,308-318], a number of multiple mutants were constructed, expressed in Escherichia coli, and assayed with androstenedione, progesterone, and benzyloxyresorufin. Simultaneous substitutions of Ile-114, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 with Phe, Thr, Val, and Ala, respectively from 2B5, resulted in a marked increase in androstenedione 15alpha- and 16alpha-hydroxylation compared with the wild-type enzyme and yielded a metabolite profile indistinguishable from that of cytochrome P450 2B5. Likewise, the reciprocal P450 2B5 quadruple mutant exhibited the specificity for 16beta-hydroxylation characteristic of the 2B4 wild type. The two reciprocal quadruple mutants of P450 2B4 and 2B5 also displayed benzyloxyresorufin dealkylase activities similar to those of the wild-type P450 2B5 and 2B4, respectively. However, the progesterone metabolite profile of P450 2B5 was not identical to that of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue. Therefore, the 17beta-acetyl group on progesterone as opposed to the oxo group on androstenedione may lead to interaction with additional amino acid residues.

Androstenedione↗
PubMed1996

Elucidation of amino acid residues critical for unique activities of rabbit cytochrome P450 2B5 using hybrid enzymes and reciprocal site-directed mutagenesis with rabbit cytochrome P450 2B4.

The molecular basis for the unique activities of rabbit cytochrome P450 2B5, compared with the highly related rabbit 2B4, was investigated using hybrid enzymes and site-directed mutagenesis. Alterations in androstenedione hydroxylase profiles observed with 2B4-2B5 hybrids expressed in COS cells showed that key amino acids are present in both the N-terminal ApaI fragment (codons 1-122) and an internal SstI fragment (codons 220-393). Based on these results, data obtained with other cytochromes P450 2B, and correlation to the six substrate recognition sites proposed by Gotoh (1992, J. Biol. Chem. 267, 83-90), reciprocal 2B4-2B5 mutants were constructed at positions 114, 294, 363, and 367. Wild-type and mutant enzymes were expressed in Escherichia coli, and the oxidation of a number of substrates was analyzed. All residues studied were found to be important for regio- and stereospecificity of androstenedione hydroxylation. Mutations at these positions also caused alterations in the oxidation of progesterone, benzyloxyresorufin, pentoxyresorufin, ethoxycoumarin, and benzphetamine, with the magnitude and direction of the changes dependent upon the enzyme, residue, and substrate. Major changes in activity were consistently observed upon mutation of residues 114 and 294 in both enzymes, and some of these alterations were interpreted with the help of a 3-D model of P450 2B4. For example, in the 2B4 Ile-114--> Phe mutant, Phe prevents androstenedione from assuming a 16 beta-binding orientation and also hinders binding of benzyloxyresorufin, leading to a loss of activity. Conversely, the presence of Phe-114 stabilizes a 16 alpha-binding orientation of androstenedione, resulting in an increase in this activity.

Amino Acid Sequence↗
PubMed1995

Escherichia coli expression of site-directed mutants of cytochrome P450 2B1 from six substrate recognition sites: substrate specificity and inhibitor selectivity studies.

Cytochrome P450 2B1 wild-type and eight site-directed mutations at positions 114, 206, 236, 302, 363, 367, and 478 have been expressed in an Escherichia coli system. Solubilized membrane preparations yielded 100-180 nmol of P450/L of culture. The metabolism of a number of substrates including androstenedione, progesterone, (benzyloxy)resorufin, pentoxyresorufin, and benzphetamine was analyzed. The E. coli-expressed enzymes displayed the same androstenedione metabolite profiles previously observed with a COS cell expression system. Several of the mutants exhibited an increased rate of progesterone hydroxylation, possibly as the result of an enlarged substrate binding pocket and increased D-ring alpha-face binding. (Benzyloxy)resorufin and pentoxyresorufin O-dealkylation by the P450 2B1 mutants exhibited activities ranging from 10% to 99% and 3% to 71% of wild-type, respectively. Interestingly, the Val-363-->Leu mutant showed markedly suppressed pentoxyresorufin but unaltered (benzyloxy)resorufin dealkylase activity. Benzphetamine N-demethylase activities ranged from 28% to 110% of wild-type. Mechanism-based inactivation of the P450 2B1 mutants showed that susceptibility to inactivation by chloramphenicol and D-erythro- and L-threo-chloramphenicol was abolished in the Val-367-->Ala mutant. The Val-363-->Leu mutant was refractory to L-threo-chloramphenicol. Studies of chloramphenicol covalent binding and metabolism by the Val-367-->Ala mutant showed that its resistance to inactivation is largely attributable to an inability to bioactivate the inhibitor. The expression of P450 2B1 wild-type and mutants in E. coli provides an excellent opportunity to study structure/function relationships by site-directed mutagenesis.

Androstenedione↗
PubMed1994

[Trans-corneal trabeculectomy].

One of the main causes in failure of fistulizing operations for glaucoma is related to conjunctival and subconjunctival cicatrization. Trans-corneal trabeculectomy is different from conventional fistulizing operations and the conjunctival incision is avoided. Passing through the cornea, partial thickness sclera and trabecula are removed. The conjunctiva is least violated during the procedures. Trans-corneal trabeculectomy was completed in 15 eyes with glaucoma. Most of filtering blebs revealed diffuse. The conjunctival inflammatory reaction was minimal with only mild hyperemia even at the first postoperative day. Intraocular pressure of 14 eyes was normal without any medication postoperatively.

Adult↗
PubMed1992

Effects of exogenous gamma-aminobutyric acid on experimental arrhythmias.

The effects of exogenous gamma-aminobutyric acid (GABA) 10 mg.kg-1 iv in preventing arrhythmias induced by drugs and ischemia were studied in mice, rats, and guinea pigs. It was found that the threshold dose of aconitine inducing arrhythmia in mice and the recovery rate to normal sinus rhythm increased significantly, ED50 of GABA was 5.4-5.8 mg.kg-1. The duration of ventricular tachycardia (VT) induced by aconitine in rats was shortened (P < 0.01). The incidence and the mortality of ventricular fibrillation (VF) in GABA group were decreased to 0/10 vs 6/10 and 5/10 in control, respectively (P < 0.05). The doses of ouabain to induce ectopic beats (EB), VT, VF, and cardiac arrest (CA) in guinea pigs were increased (P < 0.01). The incidence of VF induced by coronary artery ligation in rats was decreased to 0/5 in GABA group vs 4/5 in control group (P < 0.01). The total amount of EB, total time of VT, and VF were 66%, 41%, and 0% of the control group, respectively. The anti-arrhythmic effects of GABA were dose-dependent and as potent as procainamide (10 or 5 mg.kg-1, iv). The results suggest GABA (10 mg.kg-1, iv) may be useful for the prevention of VT and VF.

Aconitine↗
PubMed1989

[Studies of the active constituents of the Chinese drug "duhuo" Angelica pubescents].

Eight coumarins isolated from the alcohol extract of the Chinese drug "Duhuo", the root of Angelica pubescents Maxim. f. biserrata Shan et Yuan (Umbelliferae) were elucidated to be columbianetin (I), columbianetin acetate (II), columbiadin (III), osthol (IV), isoimperatorin (V), bergapten (VI), xanthotoxin (VII), and columbianetin-beta-D-glucopyranoside (VIII), by chemical and spectral analysis, compound VIII was isolated from plant for the first time. All these coumarins were tested on platelet aggregation induced by 2 microns ADP. I, II, III, IV and VIII showed strong inhibiting activity against platelet aggregation.

Animals↗