Biomedical subjects

Y K Li

Publications and source records attributed to Y K Li.

At least 19 recordsLinked to original sources

PubMed2005

Early diffusion weighted imaging and expression of heat shock protein 70 in newborn pigs with hypoxic ischaemic encephalopathy.

OBJECTIVE: To establish a cerebral hypoxia ischaemic model in piglets and to explore the early diffusion weighted imaging (DWI) as well as the expression of heat shock protein 70 (HSP70) with hypoxic ischaemic encephalopathy (HIE). METHODS: Twenty four 3 day old piglets were permanently ligated common carotid arteries bilaterally and ventilated with 3% oxygen for 30 minutes, and their brains were examined at three hours, six hours, and 24 hours after hypoxic ischaemia with T2 weighted images and diffusion weighted images. Apparent diffusion coefficient (ADC) values were measured and HSP70 expression was detected in the central and peripheral regions of lesions. RESULTS: Abnormal signal intensity was detected on DWI and ADC maps in three hypoxic ischaemic groups. ADC values in the central lesions rapidly decreased, whereas ADC values in the peripheral regions reduced prominently at 24 hours after hypoxic ischaemia. There were significant differences (p<0.01) between ADC values of the central regions and the peripheral regions of lesions. The number of HSP70 positive cells was low at three hours, raised at the highest peak at six hours, and then rapidly decreased. There was statistical difference between HSP70 positive cells in the central regions and those in peripheral regions (p<0.01). CONCLUSION: DWI may identify more early lesions, and improve conspicuity in the acute setting. ADC values can be used as an objective measure for hypoxic ischaemic injury. Tissue reversibility can be further reflected according to the information of ADC values and HSP70 immunohistochemical staining.

Animals↗

PubMed2001

Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247.

A family 3 beta-glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been cloned and overexpressed. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including substrate reactivity, secondary kinetic isotope effects and inhibition studies. The stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction. Based on the k(cat) values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behaviour is consistent with a two-step mechanism involving the formation and breakdown of a glucosyl-enzyme intermediate. The large Bronsted constant (beta=-0.85) for the leaving-group-dependent portion (pK(a) of leaving phenols >7) indicates substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl beta-D-glucopyanoside, o-nitrophenyl beta-D-glucopyanoside and p-cyanophenyl beta-D-glucopyanoside as substrates were 1.17+/-0.02, 1.19+/-0.02 and 1.04+/-0.02 respectively. These results support an S(N)1-like mechanism for the deglucosylation step and an S(N)2-like mechanism for the glucosylation step. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (k(cat)/K(m)) of the D247G and D247N mutants were 30000- and 200000-fold lower respectively than that of the wild-type enzyme, whereas the D247E mutant retained 20% of wild-type activity. These results indicate that Asp-247 is an essential amino acid. It is likely that this residue functions as a nucleophile in the reaction. This conclusion is supported by the kinetics of the irreversible inactivation of the wild-type enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mutant.

Aspartic Acid↗

PubMed2001

Rapid process for purification of an extracellular beta-xylosidase by aqueous two-phase extraction.

A rapid process for purification of an extracellular beta-xylosidase with high purity was developed. The manipulation involved the precipitation of protein from culture medium and the extraction of enzyme from the resuspended crude protein solution by an aqueous-two phase separation. A linear random copolymer, PE62, with 20% ethylene oxide and 80% propylene oxide was employed in both stages of the purification. The enzyme was precipitated effectively by using 10% (w/v) PE62 and 5% (w/v) Na2HPO4. The aqueous two-phase extraction was performed with PE62 (10%)-NaH2PO4 (15%) as phase-forming reagent. SDS-PAGE analysis revealed that the purified enzyme is near homogeneity. The yield is about 100% with a purification factor of 8.8-fold. The whole process can be completed within an hour without any column chromatography.

Chemical Precipitation↗

PubMed2001

Pulmonary haemorrhage in a patient with Kikuchi disease.

Kikuchi disease is an immunologic disease and most commonly presents with cervical lymphadenopathy. It is usually self-limiting [1]. It is sometimes associated with systemic lupus erythematosus (SLE) [2], and can be associated with many complications including aseptic meningitis, neuropathy, brachial neuritis. It can also be associated with lymphoma, and may sometimes be misdiagnosed as such [3]. The following is the first reported case of Kikuchi disease associated with pulmonary haemorrhage. The patient died and no other cause of the pulmonary haemorrhage could be identified at post mortem.

Adult↗

PubMed2000

Effective induction, purification and characterization of Trichoderma koningii G-39 beta-xylosidase with high transferase activity.

A beta-xylosidase was induced and purified from the culture filtrate of Trichoderma koningii G-39, grown in a medium containing 1% oat spelts xylan and 0.1% xylose. The presence of xylose unequivocally enhanced the induction of beta-xylosidase. The purified enzyme, which exhibited a significant alpha-arabinosidase activity, was obtained with high yield simply via ethanol precipitation and a single anion-exchange chromatography and was characterized as a monomeric glycoprotein with an estimated molecular mass of 104 kDa and a pI of 4.6. The K(m) values towards p-nitrophenyl beta-D-xylopyranoside and p-nitrophenyl alpha-L-arabinopyranoside are 0.04 and 7.5 mM, respectively. It is stable at pH 2.5-7.4, 37 degrees C. The pH and temperature optima are in the range of 3.5-4.0 and 55-60 degrees C, respectively. Contrary to most beta-xylosidases from other sources, Hg(2+) (up to 25 mM) has no effect on enzyme activity. Xylose was shown to inhibit the purified enzyme with a moderate K(i) value of 5 mM. The enzyme exhibited transxylosylation activity and was characterized as a 'retaining' enzyme, catalysing the hydrolysis of substrate with the retention of anomeric configuration.

Biotechnology↗

PubMed2000

An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides.

A chitosan-degrading fungus, designated Aspergillus sp. Y2K, was isolated from soil. The micro-organism was used for producing chitosanase (EC 3.2.1.132) in a minimal medium containing chitosan as the sole carbon source. The induced chitosanase was purified to homogeneity from the culture filtrate by concentration and cationic SP-Sepharose chromatography. The purified enzyme is a monomer with an estimated molecular mass of 25 kDa by SDS/PAGE and of 22 kDa by gel-filtration chromatography. pI, optimum pH and optimum temperature values were 8.4, 6.5 and 65-70 degrees C, respectively. The chitosanase is stable in the pH range from 4 to 7.5 at 55 degrees C. Higher deacetylated chitosan is a better substrate. Chitin, xylan, 6-O-sulphated chitosan and O-carboxymethyl chitin were indigestible by the purified enzyme. By endo-splitting activity, the chitosanase hydrolysed chitosan to form chitosan oligomers with chitotriose, chitotetraose and chitopentaose as the major products. The enzyme hydrolyses chitohexaose to form chitotriose, while the chitopentaose and shorter oligomers remain intact. The N-terminal amino acid sequence of the enzyme was determined as YNLPNNLKQIYDDHK, which provides useful information for further gene cloning of this enzyme. A 275 g-scale hydrolysis of chitosan was performed. The product distribution was virtually identical to that of the small-scale reaction. Owing to the simple purification process and high stability of the enzyme, it is potentially valuable for industrial applications.

Amino Acid Sequence↗

PubMed2000

Mechanistic study of beta-xylosidase from Trichoderma koningii G-39.

The catalytic mechanism of the beta-xylosidase purified from the culture filtrate of Trichoderma koningii G-39 was investigated. By NMR spectroscopy, the stereochemistry of the enzyme catalyzing the hydrolysis of 2,4-dinitrophenyl and p-nitrophenyl-beta-D-xylosides was found unequivocally to involve retention of the anomeric configuration. Based on the k(cat) values of a series of arylxylosides with leaving group pK(a)s in the range of 4-10, an extended Bronsted plot was constructed with a slope (beta(lg)) near zero. Enzymatic hydrolysis of aryl-beta-D-xylosides in acetate buffer (pH 4.0) containing 3 or 5% methanol showed a constant product ratio (methylxyloside/xylose), indicating the presence of a common intermediate, probably the xylosyl-enzyme intermediate. In the presence of DTT, the k(cat) values of p-cyanophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-D-xylopyranoside increased greatly. A two-step mechanism involving the formation and breakdown of the xylosyl-enzyme intermediate was therefore proposed. The rate-limiting step is the breakdown of the intermediate. The secondary deuterium kinetic isotope effect (k(H)/k(D)) measured for 2,4-dinitrophenyl-beta-D-xyloside was 1.02+/-0.01, suggesting that the transition state for breakdown of the xylosyl-enzyme intermediate is S(N)2-like.

Binding, Competitive↗

PubMed1999

Changes and implications of blood flow velocity of the vertebral artery during rotation and extension of the head.

OBJECTIVE: To study the effects of extension and extension-rotation of the head on the blood flow velocity of the vertebral artery experimentally and clinically. DESIGN: Randomized experimental clinical study. SETTING: Institute of Clinical Anatomy and Biomechanics and the Department of Ultrasound, NanFang Hospital of the First Military Medical University, Guangzhou, China. SUBJECTS: The fresh spines from T1-2 to the occipital bone were obtained from persons who died of acute brain death (n = 10). For the transcranial Doppler measurement, 27 asymptomatic subjects with routine physical examination results and 23 students from the above-mentioned university took part in the test. INTERVENTIONS: The instillation experiment in the cadavers was made. Clinical measurements of blood flow velocity in the vertebral artery in the subjects and students were taken and recorded. MAIN OUTCOME MEASURES: Changes in the drop of the vertebral artery were measured by the instillation test in the experimental sample. The blood flow velocity was measured with transcranial Doppler sonography during extension and extension-rotation of the head. RESULTS: During one-sided rotation of the head with extension of the head, measurements in the contralateral vertebral artery and the bilateral vertebral arteries were both reduced in the instillation experiment. The blood flow velocity in the bilateral vertebral arteries decreased during extension and extension-rotation of the head in the subjects and the students. The pulsatility index in the right vertebral arteries increased more than that in the left in extreme extension and that in the bilateral vertebral arteries in neutral position. CONCLUSIONS: Extreme rotation and extension are dangerous to patients who have abnormal vertebral arteries when extreme rotatory and extension manipulations are applied. Doctors should be very careful when rotating the patient's head to the right side.

Adult↗

PubMed1998

New imidazoles as probes of the active site topology and potent inhibitors of beta-glucosidase.

Series of 4-arylimidazoles, omega-N-acylhistamines and 4-(omega-phenylalkyl)imidazoles were synthesized in order to probe the active site topology of sweet almond beta-glucosidase. These imidazole derivatives were shown to be very powerful competitive inhibitors. Among the 20 tested compounds, omega-N-benzoylhistamine and 4-(3'-phenylpropyl)imidazole are the most potent inhibitors of the enzyme, with pH-independent Ki values of 0.06 and 0.07 microM, respectively. The inhibition of 4-(omega-phenylalkyl)imidazoles exhibited an interesting trend as to Ki values: 4-phenylimidazole (6.6 microM)>4-benzylimidazole (1.4 microM)>4-(2'-phenylethyl)imidazole (0.82 microM)>4-(3'-phenylpropyl)imidazole (0.07 microM)<4-(4'-phenylbutyl)imidazole (0.13 microM)<4-(5'-phenylpentyl)imidazole (0.3 microM). This revealed that the imidazole and aryl binding sites (which result from favorable interactions within the corresponding glycone and aglycone binding subsites) are separated by the optimal distance equivalent to the length of a -CH2-CH2-CH2- group. Substitutions of the phenyl moieties of 4-phenylimidazole and 4-benzoylhistamine result in weaker inhibition. These classes of imidazoles are particularly powerful inhibitors of sweet almond beta-glucosidase.

Binding Sites↗

PubMed1998

The effect of cervical traction combined with rotatory manipulation on cervical nucleus pulposus pressures.

OBJECTIVE: To study experimentally the effect of cervical traction combined with rotatory manipulation on the pressure of the cervical nucleus pulposus. DESIGN: Randomized experimental study. SETTING: Institute of Clinical Anatomy and Biomechanics, the First Military Medical University, GuangZhou, China. SUBJECTS: Twelve fresh cervical spines, from C7 to occipital bone, were obtained from cadavers of patients who had died from acute brain death. INTERVENTION: State A: under different traction forces, the cervical spine was rotated. State B: the cervical spine was rotated first and then tractioned. State C: the cervical spine was tractioned and rotated simultaneously. MAIN OUTCOME MEASURES: The cervical spine was tractioned and rotated by the Material Test System (MTS), and pressures of the cervical nucleus pulposus of C3-4, C4-5 and C5-6 were measured using pressure sensors. RESULTS: (a) When the traction force increased, the pressure fell continuously in the 200-N tractioned spines; the pressure increased slightly when the sample was rotated. (b) The pressure fell to a certain extent when the state of cervical spine was restored. (c) In state A, the pressure fell obviously and increased slightly under a 200-N traction force and then the sample was rotated; in state B, the pressure first increased to a certain extent and then fell slightly and in state C, the pressure underwent no change in the main. CONCLUSIONS: The results of this research suggest that rotatory manipulation of cervical spine under traction was the safest of the three procedures and the traction force used in clinical treatment may be a little smaller.

Cervical Vertebrae↗

PubMed1997

[The epidemiologic value of polymerase chain reaction to the diagnosis of brucellosis patients].

PCR was used to test fifty-four brucellasis patients and thirty-six healthy people as controls, who were all from Man Tang Chuan and Yi He communities, Sui De county, Shan Xi Province, where brucellosis out-break was occurred in 1996. The diagnostic titres of SAT for brucellasis patients was set 1:100++. The positive rates of PCR in patients and controls were 75% and 65.7% respectively, comparing with the positive rates of RBPT 96.3% and 30.6% in the same two groups. At the same time, we also detected eight people who had received immunization one month to thirty-two years ago, with positive rates for PCR and RBPT 100% and 87.5% respectively. The results showed that there was no significant diversity of PCR results among acute, subacute and chronic brucellosis patients. It was revealed that the serological results were related to the course of disease, but the results came out of PCR method which was used to detect target DNA was not influenced by the course of disease.

Animals↗

PubMed1997

[Clinical studies on liangxue piyan granule no. 2 in treating psoriasis].

OBJECTIVE: To observe the effect of Liangxue Piyan granule No. 2 (LPG) on psoriasis. METHODS: Ninety-six cases of psoriasis patients were treated with LPG and a control group of 32 cases was set up, which was treated with Fufang Qingdai Capsule (FQC). RESULTS: The total effective rate of LPG was 84.7%, that of the control group was 71.9% (P > 0.05), there was no significant difference between two drugs in treatment of psoriasis. The recurrent rate of LPG group (3.6%) was lower than that of control (21.7%), P < 0.01. CONCLUSION: This granule provides a better drug for treatment of psoriasis.

Adolescent↗

PubMed1995

Ultraviolet spectroscopic evidence for decreased motion of the active site tyrosine residue of delta 5-3-ketosteroid isomerase by steroid binding.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the highly efficient conversion of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a stereoselective and intramolecular transfer of the 4 beta-proton to the 6 beta-position. Tyr-14 is the critical general acid and Asp-38 is the general base involved in catalysis. The UV absorption bandwidths of Tyr-14 were much narrower than those of the other two tyrosines (Tyr-55 and Tyr-88) of isomerase or of the N-acetyltyrosine ethyl ester in aqueous solution, suggesting that Tyr-14 is restricted in its mobility. Further immobilization of this residue occurs upon steroid binding. Thus, 5 alpha-estrane-3,17-dione, an A-ring saturated steroid, induces significant narrowing of the tyrosine absorption bands (pi-->pi*) of the main peak (279.5 nm) and the shoulder (285.5 nm) of Tyr-14, with no significant changes in lambda max. No effects of steroid binding were found on the absorption bandwidths of Tyr-55, Tyr-88, or the phenylalanine residues. The ratio of the absorbance (Amax) at the absorption maximum (lambda max) to that at lambda max plus 4 nm (Amax +4) was used as a measure of peak sharpness. Specifically, the ratios of A279.5/A283.5 (main peak) and A285.5/A289.5 (shoulder) of Tyr-14 of the free enzyme at 25 degrees C were 1.25 and 1.89, respectively, and they increased to 1.41 and 2.70, respectively, in the complex. A more precise measurement of the band narrowing from 4.2 to 3.1 nm between the inflection points was obtained from the derivative spectra. The absorption bands of free and steroid-bound isomerase were narrowed significantly by lowering the temperature and were broadened by denaturation, suggesting that the unusual peak-sharpening effects induced by steroid binding arise from the restricted motion of Tyr-14, as well as from more directional hydrogen bonding resulting from the displacement of water molecules from the active site and decreased flexibility of the protein. Larger enthalpy of the sharpening effects was observed for the steroid-bound enzyme (-0.527 +/- 0.016 kcal/mol) than for the free enzyme (-0.250 +/- 0.018 kcal/mol) by lowering the temperature, indicating that interactions of Tyr-14 with its environment, which restrain its motion, are stronger in the steroid-bound enzyme than in the free enzyme. Hydrogen-bonding modes of Tyr-14, mobility of the active site, and protein flexibility are the environmental factors determining the absorption bandwidths of the critical Tyr-14 residue.

Binding Sites↗

PubMed1994

Characterization of the three tyrosine residues of delta 5-3-ketosteroid isomerase by time-resolved fluorescence and circular dichroism.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids via an enolic intermediate. Site-specific mutagenesis has identified Tyr-14 and Asp-38 as the catalytically essential general acid and base, respectively. Three tyrosine residues (Tyr-14, Tyr-55, and Tyr-88) are the only significant fluorophores in the wild-type isomerase. Recent studies of the steady-state fluorescence of the wild-type enzyme and all six mutant enzymes in which one or two tyrosine residues have been mutated to phenylalanine show that the fluorescence intensity of Tyr-14 is very high, that of Tyr-88 is very low, and that of Tyr-55 is intermediate and comparable to that of N-acetyltyrosine amide in solution (Li, Y.-K., Kuliopulos, A., Mildvan, A.S., & Talalay, P. (1993) Biochemistry 32, 1816-1824). Extension of these experiments by time-resolved fluorescence and fluorescence anisotropy measurements demonstrates that Tyr-14, which is in a hydrophobic environment, has an unusually long fluorescence lifetime (4.6 ns) as compared to Tyr-55 (2.0 ns) or Tyr-88 (0.8 ns) and to most protein tyrosine residues (0.2-2 ns). The Förster distances obtained from the absorption and emission of these tyrosines predict that total quenching of Tyr-14 fluorescence by Tyr-55, and to a lesser degree by Tyr-88, would occur if their orientations were favorable.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗

PubMed1994

[The morphological changes in trabecular architecture of bone in severe primary hyperparathyroidism].

By histomorphometry bone biopsy specimens were evaluated from 10 patients with severe primary hyperparathyroidism (PHPT). The data suggest that the mean trabecular width and trabecular bone volume were slightly, but not significantly increased. As indices of bone remodeling activity, both trabecular osteoid surface and trabecular resorption surface were significantly increased indeed, indicating high bone turn-over occurred in PHPT. The dynamic study showed that much more trabecular bone surface was covered either with single or double tetracycline labels in PHPT than in control. The overproduction of osteoid in the patients leads to the accumulation of osteoid on the trabecular surface. Moreover, the mean osteoid width seemed to be thicker in PHPT than in control, suggesting the co-existence of osteomalacia, which might be a possible explanation for eucalcemia usually seen in the majority. Part of chinese PHPT patients.

Adenoma↗

PubMed1994

[Trabecular connectivity in primary hyperparathyroidism].

By bone histomorphometry, 10 cases of primary hyperparathyroidism (PHPT) were used to study the trabecular connectivity, which in a few literatures was mentioned to be reserved somehow. Our data suggest that in comparison with the normal control (10 subjects) the nodes (N.Nd), the number of terminus (N.Tm), the total strut length (TSL), the length between the nodes (Nd.Nd), trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th) were significantly increased while no significance was found in N.Nd/N.Tm, Tm.Tm and TBV. The correlation analyses indicate that there is no correlation between the variables in the normal controls. In contrast, however, TBV and Tb.Th showed negative correlations with N.Tm and Tm.Tm. Besides these, there were positive correlations between N.Nd/N.Tm and TBV or Tb.Th, and a negative correlation between N.Nd/N.Tm and Tb.Sp. The results suggest that to certain degree the trabecular connectivity in PHPT be destructed while the three dimensional architecture be reserved. Our data seem to be different from those reported previously. It may be due to the severity of PHPT in our patients.

Adolescent↗

PubMed1993

Phosphonate inhibitors of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase.

Several bisphosphonates were examined as inhibitors of yeast GPD (glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12) and PGK (phosphoglycerate kinase, EC 2.7.2.3). The phosphonomethyl analog of 2-deoxy-1,3-bisphosphoglycerate (i.e., 2-oxo-1,5-bisphosphonopentane, 2-oxo-PC5P) is a good inhibitor of PGK (Ki = 0.2 +/- 0.08 mM at pH 8.5, 27 degrees C) and a poor inhibitor of GPD (Ki = 20 +/- 1 mM, pH 8.5). The shorter, butane, analog (2-oxo-PC4P) binds more tightly to PGK (Ki = 84 +/- 6 microM), and about equally well to GPD, as does 2-oxo-PC5P. The 2-oxo-bisphosphonates bind to PGK more tightly (by approx. 4 kJ/mol) than do the corresponding non-carbonyl analogues (1,4-bisphosphonobutane and 1,5-bisphosphonopentane).

Diphosphoglyceric Acids↗

PubMed1993

Environments and mechanistic roles of the tyrosine residues of delta 5-3-ketosteroid isomerase.

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids by a stereoselective and conservative transfer of the 4 beta-proton to the 6 beta-position. The 10(9.5)-fold enzymatic rate acceleration can be attributed to a concerted rate-limiting enolization in which Tyr-14 and Asp-38, positioned orthogonally, act as general acid and base, respectively. The pKa value of the phenolic hydroxyl group of Tyr-14 of the Y55F/Y88F double mutant is 11.6 +/- 0.2 by UV titration. However, the fluorescence titration of Tyr-14 shows biphasic sigmoidal behavior with apparent pKa values of 9.5 and 11.5. This suggests the assistance of a basic residue at the active site, possibly a lysine or tyrosine residue. Mutation of each of the four lysine residues K119L, K108Q, K92Q, and K60Q lowered specific activities only slightly to between 43% and 98% of the wild-type enzyme. Similarly, mutations of Tyr-55, Tyr-88, or both to phenylalanine led to only 2-4-fold reductions in catalytic activity. These findings suggest that despite the enormous difference between the pKa value of Tyr-14 (11.6) and that of the 3-carbonyl group of the steroid (ca. pKa-7), the reaction may rely on the concerted participation of Tyr-14 and Asp-38 only. The apparent pKa value of 9.5 in the fluorescence titration of Tyr-14 and in kinetic measurements probably results from conformational changes of the enzyme. The unusually high pKa value of Tyr-14 of 11.6 +/- 0.2 was used to estimate a local dielectric constant of 18 +/- 2 near this residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗