Biomedical subjects

X M Pan

Publications and source records attributed to X M Pan.

At least 19 recordsLinked to original sources

PubMed2001

Cysteine-25 of adenylate kinase reacts with dithiothreitol to form an adduct upon aging of the enzyme.

Adenylate kinase (AK) ages in solution in the presence of DL-dithiothreitol (DTT) with a gradual activity decrease. Upon dilution with 4 M guanidine hydrochloride denatured native and aged AK, both recover to the same activity as the fresh enzyme. Mass spectroscopy and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole chloride modification kinetics studies identify that the residue cysteine-25 of the enzyme reacts with DTT to form an adduct. The formation of the unusual bridging DTT adduct of AK appears to be the result of a stable DTT-protein complex. The K(M) for AMP, ADP and MgATP of the DTT-modified enzyme does not differ significantly from that of the intact enzyme, whereas the secondary and tertiary structures of the enzyme change obviously. These results indicate that cysteine-25 may not be involved directly in substrate binding, but may play an important role in maintaining secondary and tertiary structures of native AK, as well as the conformation interconversion in the catalytic cycle.

Adenylate Kinase↗

PubMed2001

Multiple linear regression for protein secondary structure prediction.

In the present work, a novel method was proposed for prediction of secondary structure. Over a database of 396 proteins (CB396) with a three-state-defining secondary structure, this method with jackknife procedure achieved an accuracy of 68.8% and SOV score of 71.4% using single sequence and an accuracy of 73.7% and SOV score of 77.3% using multiple sequence alignments. Combination of this method with DSC, PHD, PREDATOR, and NNSSP gives Q3 = 76.2% and SOV = 79.8%.

Databases, Factual↗

PubMed2001

New method for accurate prediction of solvent accessibility from protein sequence.

A novel method was developed for predicting the solvent accessibility. Based on single sequence data, this method achieved 71.5% accuracy with a correlation coefficient of 0.42 in a database of 704 proteins with threshold of 20% for a two-state-defining solvent accessibility. Prediction in a data subset of 341 monomeric proteins achieved 72.7% accuracy with a correlation coefficient of 0. 43. On the average, prediction over short chains gives better results than that over long chains. With a solvent accessibility threshold of 20%, prediction over 236 monomeric proteins with chain length < 300 amino acid residues achieved 75.3% accuracy with a correlation coefficient of 0.44 by jackknife analysis, which is higher than that obtained by previous methods using multiple sequence alignments.

Algorithms↗

PubMed2001

Accurate prediction of protein secondary structural content.

An improved multiple linear regression (MLR) method is proposed to predict a protein's secondary structural content based on its primary sequence. The amino acid composition, the autocorrelation function, and the interaction function of side-chain mass derived from the primary sequence are taken into account. The average absolute errors of prediction over 704 unrelated proteins with the jackknife test are 0.088, 0.081, and 0.059 with standard deviations 0.073, 0.066, and 0.055 for alpha-helix, beta-sheet, and coil, respectively. That the sum of predicted secondary structure content should be close to 1.0 was introduced as a criterion to evaluate whether the prediction is acceptable. While only the predictions with the sum of predicted secondary structure content between 0.99 and 1.01 are accepted (about 11% of all proteins), the absolute errors are 0.058 for alpha-helix, 0.054 for beta-sheet, and 0.045 for coil.

Algorithms↗

PubMed2000

Both native conformers of rabbit muscle adenylate kinase are active.

There are two forms of rabbit muscle adenylate kinase (AK) with different 8-anilino-1-naphthalenesulfonic acid (ANS) binding properties in equilibrium solution. One form (about 70%, denoted N1) binds rapidly with ANS, whereas the other (about 30%, denoted N2) does not. Furthermore, native forms of AK should adopt different conformations for binding with substrates and products, which should be pre-existing for performing its catalytic function. The present experiments demonstrate both forms of AK distinguished by ANS probe are active. The activity of N2 is about 0.8 fold higher than N1 and shows higher susceptibility to proteolysis by trypsin. This means that the native state of AK might be an ensemble of kinetically attainable conformers and the energy landscapes of AK folding should be rugged with more than one local minimum.

Adenylate Kinase↗

PubMed2000

Atheroemboli to the brain: size threshold for causing acute neuronal cell death.

OBJECTIVE: The objective of this study was to investigate the dilemma posed by the observations that carotid angioplasty dislodges significant numbers of plaque fragments but is reported to have a low rate of neurologic consequences. We examined the fragments released by ex vivo carotid angioplasty. The smaller and most numerous were separated by size and injected into rats to determine the tolerance of the brain to microemboli. METHODS: Ex vivo angioplasty was performed on a total of 20 human carotid plaques removed en bloc. Plaques were placed within polytetrafluoroethylene grafts, and three manipulations were performed: guide wire insertion, 3.5- or 4.0-mm balloon angioplasty, and 5-mm angioplasty with or without a Palmaz stent. After each manipulation, the lumen was flushed, effluent was collected, and fragments were counted under 100x magnification. Using 200-microm and 500-microm micropore mesh, we separated fragments by size into two groups: (1) less than 200 microm and (2) 200 to 500 microm. We then injected rats with saline alone (Group A), with 100 fragments less than 200 microm (Group B), or with 100 fragments 200 to 500 microm (Group C). Animals were euthanized at 1, 3, and 7 days, and brain sections were examined for cell viability and expression of HSP- 72. RESULTS: The total number of fragments dislodged from the plaques varied from 30 to 553. The mean number of fragments released with each manipulation was as follows: guide wire passage, 24; initial balloon angioplasty, 97; second balloon angioplasty, 68; and second angioplasty plus stent, 172. Sixteen of the 20 plaques dislodged fragments that were 1 mm or more in greatest dimension. There was no evidence of brain ischemia in Group A at any time. Group B also showed no injury at 1 or 3 days. However, injection of 200- to 500-microm fragments (Group C) caused a scattered pattern of neuronal cell death. At 7 days, brain sections from both Group B and Group C animals had a scattered pattern of ischemic neurons. There were no classic wedge-shaped infarctions. DISCUSSION: The brain appears to have a surprising tolerance for microembolization in the acute setting. Thus, carotid angioplasty may dislodge plaque fragments, but there may still be a low incidence of stroke. However, even small plaque fragments, less than 200 microm, may cause neuronal ischemia at later time points. Periprocedural microemboli could cause subtle neurologic dysfunction in late follow-up.

Angioplasty, Balloon↗

PubMed2000

Oxidized cholesterol in the diet accelerates the development of atherosclerosis in LDL receptor- and apolipoprotein E-deficient mice.

The aim of the current study was to determine whether oxidized cholesterol in the diet accelerates atherosclerosis in low density lipoprotein receptor- (LDLR) and apolipoprotein E- (apo E) deficient mice. Mice were fed either a control diet or a diet containing oxidized cholesterol. For LDLR-deficient mice, the control diet consisted of regular mouse chow to which 1.0% cholesterol was added. The oxidized diet was identical to the control diet except that 5% of the added cholesterol was oxidized. In apo E-deficient mice, the control diet contained 0.15% cholesterol, whereas in the oxidized diet, 5% of the added cholesterol was oxidized. LDLR-deficient and apo E-deficient mice were fed the experimental diets for 7 and 4 months, respectively. In mice fed the oxidized-cholesterol diets, the levels of oxidized cholesterol in sera were increased. At the end of the experiment, aortas were removed and atherosclerosis was assessed. We found that in LDLR-deficient mice, feeding of an oxidized-cholesterol diet resulted in a 32% increase in fatty streak lesions (15.93+/-1.59% versus 21.00+/-1.38%, P<0.03). Similarly, in apo E-deficient mice, feeding of an oxidized-cholesterol diet increased fatty streak lesions by 38% (15.01+/-0.92% versus 20. 70+/-0.86%, P<0.001). The results of the current study thus demonstrate that oxidized cholesterol in the diet accelerates fatty streak lesion formation in both LDLR- and apo E-deficient mice.

Animals↗

PubMed1999

An iso-random Bi Bi mechanism for adenylate kinase.

An iso-random Bi Bi mechanism has been proposed for adenylate kinase. In this mechanism, one of the enzyme forms can bind the substrates MgATP and AMP, whereas the other form can bind the products MgADP and ADP. In a catalytic cycle, the conformational changes of the free enzyme and the ternary complexes are the rate-limiting steps. The AP(5)A inhibition equations derived from this mechanism show theoretically that AP(5)A acts as a competitive inhibitor for the forward reaction and a mixed noncompetitive inhibitor for the backward reaction.

Adenosine Monophosphate↗

PubMed1999

What is the minimum number of residues to determine the secondary structural state?

The failure of protein secondary structural prediction is commonly attributed to the neglect of long-range interactions. The question is, what is the minimum length of subsequence required to determine the central secondary structural state, stabilized only by local interactions? In the present work, the 20 amino acids were classified into eight groups to analyze systematically the relationship between the length and secondary structural state of subsequences in the PDB database. It was found that the fraction of subsequences with a unique central secondary structural state increases with increasing length, and the minimum length of subsequence required to determine the central secondary structural state is about 14-17 residues. The low accuracy of secondary structure prediction does not result from the neglect of long-range interactions, but may result from the limitation of the available protein database size or prediction algorithm.

Algorithms↗

PubMed1999

A novel method for increasing production of mature proteins in the periplasm of Escherichia coli.

A novel strategy to obtain high-level production of mature proteins exported to the periplasm of Escherichia coli is described. It is based on a modified signal sequence generated by insertion of a coding sequence of the polypeptide precursor of interest at the BamHI site of the commercial vector pQE-30 resulting in an addition of a dodeca-peptide (MRGSH6GS) at the N-terminus of the precursor. The modification does not affect correct processing of the modified signal nor proper folding of the target protein, resulting in an untagged native product. The method is simple for avoiding onerous optimization of translation initiation and screening of host stains. The usefulness of this method is illustrated by overexpression of DsbC and DsbA. Induced by 0.01 mM IPTG at 37 degrees C, proteins were overproduced to comprise 20-30% of the total cellular proteins, and more than 95% of the expressed proteins were correctly processed and exported into the periplasm with yields of more than 100 mg per liter culture.

Amino Acid Sequence↗

PubMed1999

Effect of oxidized lipids in the diet on oxidized lipid levels in postprandial serum chylomicrons of diabetic patients.

OBJECTIVE: To determine whether humans with type 2 diabetes have increased levels of oxidized fatty acids in their serum chylomicron fraction after the ingestion of dietary oxidized fatty acids. RESEARCH DESIGN AND METHODS: The study was performed on 31 male type 2 diabetic patients and 24 age-matched control subjects. Among the diabetic patients, 22 had poor glycemic control, defined as HbA1 > 10% (normal value < 7.7%). Nine patients had good glycemic control (HbA1 < or = 10). Heated corn oil containing low or high levels of oxidized fatty acids was used as a test meal. At 2.5 h after the test meal, 50-ml blood samples were obtained from all subjects, and the chylomicron fraction (Sf > 1,000) was isolated. The degree of oxidation in chylomicrons was determined by measuring conjugated dienes. For determining the postprandial levels of triglycerides and of oxidized lipids in serum chylomicrons over an extended time period, blood samples were obtained at 0, 2.5, 5.0, and 7.5 h for isolation of chylomicrons and determination of fatty acid oxidation. RESULTS: We found that at 2.5 h after the consumption of the test meal containing either a low or high oxidized fatty acid content, conjugated dienes in serum chylomicrons in diabetic subjects in poor glycemic control were increased compared with those in control subjects. Diabetic patients in good glycemic control had similar levels of oxidized lipid in their chylomicrons when compared with control subjects. Additionally, in diabetic patients in poor glycemic control, the levels of oxidized lipids in chylomicrons remained elevated for an extended post-prandial period. CONCLUSIONS: In diabetic subjects with poor glycemic control, dietary oxidized lipids induce an exaggerated and sustained increase in the levels of oxidized lipids in chylomicrons when compared with either control subjects or diabetic patients with good glycemic control. These increased postprandial levels of potentially atherogenic oxidized lipids may contribute to the accelerated atherosclerosis associated with diabetes.

Blood Glucose↗

PubMed1998

Refolding of urea-denatured adenylate kinase.

The refolding of urea-denatured adenylate kinase (EC 2.7.4.3) has been followed by formation of the secondary structure, change of surface hydrophobicity and recovery of catalytic activity. During refolding of adenylate kinase with a 20-80-fold dilution of 4 M urea-denatured enzyme at 10 degrees C, the formation of the secondary structure is a fast process with a rate constant of >0.16 s-1. Transient enhancement of the 8-anilino-1-naphthalenesulphonate (ANS) fluorescence intensity is followed by a fluorescence decrease to the level equal to the value characteristic of native enzyme. The desorption of ANS binding fluorescence is relatively slow and can be fitted to a first order reaction with a rate constant of 0.004 s-1 when the ANS is present in the dilution buffer. The desorption of ANS-binding fluorescence is accelerated in the presence of nucleotide substrates. The rate constants are increased to 0.049, 0. 029, 0.028 and 0.029 s-1 in the presence of 1 mM AMP, MgATP, ATP and ADP respectively. The refolding rate constant calculated from the initial fluorescence intensity after mixing ANS with protein at different refolding intervals is 0.016 s-1, which is faster than those obtained when ANS is present throughout the refolding process, indicating that the binding of ANS with a partially folded intermediate retards its further refolding to its native structure. The reactivation rate is even faster than the rates of refolding monitored in the absence of substrates, showing that the refolding is accelerated in the presence of the substrates. A possible refolding pathway and the accelerating effect of substrates are discussed.

Adenosine Diphosphate↗

PubMed1998

Evidence for at least two native forms of rabbit muscle adenylate kinase in equilibrium in aqueous solution.

The time course of 8-anilino-1-naphthalenesulfonic acid (ANS) binding to adenylate kinase (AK) is a biphasic process. The burst phase ends in the dead-time of the stopped-flow apparatus (about 15 ms), whereas the slow phase completes in about 10 min. A Job's plot tests of the binding stoichiometry demonstrates that there is one ANS binding site on AK, but only about 70% of the enzyme can rapidly bind with ANS, indicating that the conformation of native AK molecules is not homogeneous. Further kinetic analysis shows that the effects of ANS and substrates concentration on the burst and slow phase fluorescence building agree well with the multiple native forms mechanism. One form (denoted N1) binds with ANS, whereas the other (denoted N2) does not. ANS binding to N1 results in a burst phase fluorescence increase, followed by the interconversion of N2 to N1, to give the slow phase ANS binding. Under urea denaturation conditions, N2 is easily perturbed by urea and unfolds completely at low denaturant concentrations, whereas N1 is relatively resistant to denaturation and unfolds at higher denaturant concentrations. The existence of multiple native forms in solution may shed some light on the interpretation of the enzyme catalytic mechanism.

Adenylate Kinase↗

PubMed1998

Oxidized cholesterol in the diet accelerates the development of aortic atherosclerosis in cholesterol-fed rabbits.

Oxidized lipoproteins may play a role in atherosclerosis. Recently, we have demonstrated that the levels of oxidized fatty acids in the circulation correlate directly with the quantity of oxidized fatty acids in the diet and that dietary oxidized fatty acids accelerate atherosclerosis in rabbits. The present study tests the hypothesis that oxidized cholesterol in the diet accelerates the development of atherosclerosis. Rabbits were fed a diet containing 0.33% nonoxidized cholesterol (control diet) or the same diet containing 0.33% cholesterol of which 5% was oxidized (oxidized diet). Serum cholesterol levels increased to a similar extent in both groups, with the majority of cholesterol in the beta-VLDL fraction. Moreover, in the serum beta-VLDL fraction and liver, there was a significant increase in the oxidized cholesterol levels. Most importantly, feeding a diet enriched in oxidized cholesterol resulted in a 100% increase in fatty streak lesions in the aorta. Western diets contain high concentrations of oxidized cholesterol products, and our results suggest that these foods may be a risk factor for atherosclerosis.

Animals↗

PubMed1997

Activation of adenylate kinase by denaturants is due to the increasing conformational flexibility at its active sites.

The unfolding of adenylate kinase in urea or guanidine hydrochloride solutions was measured by UV absorbance at 287 nm, circular dichroism at 222 nm and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence. At concentrations less than 1.8 M of urea, the secondary and tertiary structures of AK were not noticeably perturbed. In contrast, the activity of the enzyme underwent significant changes, increasing about 1.6-fold when the urea concentration was increased to 1 M. The enzyme activity then decreased with further increases of the urea concentration. We also observed that the kinetics of ANS binding to AK by fluorescence was biphasic. The fast phase completed within the dead-time of the stopped-flow apparatus used, while the slow phase ended in about 10 minutes. The slow phase fluorescence rate constants increased from 0.0073 s-1 in the absence of denaturants to 0.0100 s-1 (about 1.4-fold) at 1 M urea and then decreased at higher urea concentrations. Similar results were obtained when guanidine hydrochloride was used as a denaturant. The change of the enzyme activity coincided with that of the rate of ANS binding during denaturation by low concentration of denaturants, suggesting that the activation of AK by denaturants may be due to the increasing conformational flexibility at its active site.

Adenylate Kinase↗

PubMed1997

Domain movement in rabbit muscle adenylate kinase might involve proline isomerization.

The fluorescence probe, 8-anilino-1-naphthalenesulfonic acid (ANS), was used to monitor the induced-fit conformational movement in rabbit muscle adenylate kinase. In 50 mM Tris-HCl buffer (pH 8.1), the time course of ANS binding to rabbit muscle adenylate kinase is a biphasic process. The fast phase completes within the dead-time of the stopped-flow equipment used (about 15 ms), while the slow phase ends in about 10 minutes. In the presence of 2.0 microM peptidyl prolyl cis/trans-isomerase, the rate constant of the slow phase reaction is accelerated about 2.4-fold, suggesting that the domain movement during ANS binding to rabbit muscle adenylate kinase may involve proline isomerization. The activation energy of the slow phase was determined to be 74.6 kJ/mol, which is comparable to the activation energy of proline cis/trans-isomerization (about 80 kJ/mol).

Adenylate Kinase↗

PubMed1997

Probing subtle acid-induced conformational changes of ribonuclease A by electrospray mass spectrometry.

The newly developed technique, electrospray mass spectrometry, was used to probe subtle conformational changes of bovine pancreatic RNase A during acid denaturation. In a dilute acid solution of pH 2.6, RNase A lost nearly all of its activity, whereas its intrinsic fluorescence intensity at 304 nm and its ellipticity at 222 nm were fairly resistant to denaturation by acetic acid. The observed maximum charged state of the enzyme in electrospray mass spectra was increased from 11+ (at pH 3.3) to 14+ (at pH 2.6). This could result from exposure of the buried basic amino acid residues R-10, K-41, H-12 and perhaps H-48.

Animals↗

PubMed1997

A blinded trial of local recombinant tissue factor pathway inhibitor versus either local or systemic heparin in a vein bypass model.

PURPOSE: Tissue factor pathway inhibitor (TFPI), an endogenous protease, is a potent inhibitor of the extrinsic pathway of coagulation. To determine whether TFPI could be used as an alternative to systemic heparin and dextran in vein bypass grafting procedures, we compared the efficacy of these agents in a blinded trial using a pig model of lower extremity vein bypass grafting. METHODS: Yorkshire pigs (60 to 75 kg) were divided into four groups of five each: systemic heparin (5 ml 10(3) U heparin, 50 ml intravenous dextran, and 10 U heparin/ml flush), local heparin (5 ml saline solution, 50 ml intravenous dextran, and 10 U heparin/ml flush), recombinant TFPI (rTFPI) (5 ml saline solution, 50 ml intravenous saline, and rTFPI 90 micrograms/ml flush), and control (5 ml and 50 ml intravenous saline and intravenous phosphate-buffered saline solution flush). The pigs were anesthetized and the lesser saphenous vein was harvested and reversed to construct a bypass from the common femoral artery to the saphenous artery at the hock. Each pig received two intravenous infusions before cross-clamping, and the artery and vein were flushed locally according to the protocol for each treatment group. Coagulation parameters were drawn 30 minutes after cross-clamping. The surgical team was blinded as to the pigs' treatment group throughout the protocol. RESULTS: The time from initial infusion until bypass completion averaged 80 minutes. Conduit patency rates at 7 days were as follows: four of five in the rTFPI group, three of five in the systemic heparin group, one of five in the local heparin group, and zero of five in the control group. The activated partial thromboplastin time was elevated (50.1 +/- 13.8 seconds) with systemic heparin but not in the other groups. CONCLUSIONS: Local administration of TFPI prevents thrombosis as effectively as systemic heparin and dextran and is superior to local heparin flush plus dextran (p = 0.02). Thus local TFPI offers an excellent alternative to systemic heparin plus dextran and avoids the risks of systemic anticoagulation.

Animals↗