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J Ding

Publications and source records attributed to J Ding.

At least 127 records · Page 7Linked to original sources
PubMed1999

Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids.

An important component of triple-drug anti-AIDS therapy is 2', 3'-dideoxy-3'-thiacytidine (3TC, lamivudine). Single mutations at residue 184 of the reverse transcriptase (RT) in HIV cause high-level resistance to 3TC and contribute to the failure of anti-AIDS combination therapy. We have determined crystal structures of the 3TC-resistant mutant HIV-1 RT (M184I) in both the presence and absence of a DNA/DNA template-primer. In the absence of a DNA substrate, the wild-type and mutant structures are very similar. However, comparison of crystal structures of M184I mutant and wild-type HIV-1 RT with and without DNA reveals repositioning of the template-primer in the M184I/DNA binary complex and other smaller changes in residues in the dNTP-binding site. On the basis of these structural results, we developed a model that explains the ability of the 3TC-resistant mutant M184I to incorporate dNTPs but not the nucleotide analog 3TCTP. In this model, steric hindrance is expected for NRTIs with beta- or L- ring configurations, as with the enantiomer of 3TC that is used in therapy. Steric conflict between the oxathiolane ring of 3TCTP and the side chain of beta-branched amino acids (Val, Ile, Thr) at position 184 perturbs inhibitor binding, leading to a reduction in incorporation of the analog. The model can also explain the 3TC resistance of analogous hepatitis B polymerase mutants. Repositioning of the template-primer as observed in the binary complex (M184I/DNA) may also occur in the catalytic ternary complex (M184I/DNA/3TCTP) and contribute to 3TC resistance by interfering with the formation of a catalytically competent closed complex.

Allosteric Regulation↗
PubMed1999

Involvement of receptor aggregation and reactive oxygen species in osmotic stress-induced Syk activation in B cells.

Syk has been shown to be activated by osmotic stress, however, the mechanisms involved are largely unknown. In this study, we demonstrated that cell shrinkage, rather than osmolarity, was responsible for osmotic stress-induced Syk activation. Osmotic stress-induced Syk activation depended partly upon aggregation of surface receptors. Moreover, intracellular reactive oxygen species were involved in mediating osmotic stress-induced Syk activation, with osmotic stress-induced Syk activation being inhibited by the pretreatment of cells with N-acetyl-cysteine and reduced glutathione. When cells were treated with the combination of sodium chloride and hydrogen peroxide, there was a synergistic activation of Syk. In conclusion, osmotic stress-induced Syk activation required suramin-inhibitable surface receptor aggregation and accumulation of intracellular reactive oxygen species.

Acetylcysteine↗
PubMed1999

Effect of pacing chamber and atrioventricular delay on acute systolic function of paced patients with congestive heart failure. The Pacing Therapies for Congestive Heart Failure Study Group. The Guidant Congestive Heart Failure Research Group.

BACKGROUND: Previous studies of pacing therapy for dilated congestive heart failure (CHF) have not established the relative importance of pacing site, AV delay, and patient heterogeneity on outcome. These variables were compared by a novel technique that evaluated immediate changes in hemodynamic function during brief periods of atrial-synchronous ventricular pacing. METHODS AND RESULTS: Twenty-seven CHF patients with severe left ventricular (LV) systolic dysfunction and LV conduction disorder were implanted with endocardial pacing leads in the right atrium and right ventricle (RV) and an epicardial lead on the LV and instrumented with micromanometer catheters in the LV, aorta, and RV. Patients in normal sinus rhythm were stimulated in the RV, LV, or both ventricles simultaneously (BV) at preselected AV delays in a repeating 5-paced/15-nonpaced beat sequence. Maximum LV pressure derivative (LV+dP/dt) and aortic pulse pressure (PP) changed immediately at pacing onset, increasing at a patient-specific optimal AV delay in 20 patients with wide surface QRS (180+/-22 ms) and decreasing at short AV delays in 5 patients with narrower QRS (128+/-12 ms) (P<0.0001). Overall, BV and LV pacing increased LV+dP/dt and PP more than RV pacing (P<0.01), whereas LV pacing increased LV+dP/dt more than BV pacing (P<0.01). CONCLUSIONS: In this population, CHF patients with sufficiently wide surface QRS benefit from atrial-synchronous ventricular pacing, LV stimulation is required for maximum acute benefit, and the maximum benefit at any site occurs with a patient-specific AV delay.

Aged↗
PubMed1999

Advances in CE/MS.

Explore the source record for details and available documents.

Chromatography, Liquid↗
PubMed1999

Thermodynamic criterion for the conformation of P1 residues of substrates and of inhibitors in complexes with serine proteinases.

Eglin c, turkey ovomucoid third domain, and bovine pancreatic trypsin inhibitor (Kunitz) are all standard mechanism, canonical protein inhibitors of serine proteinases. Each of the three belongs to a different inhibitor family. Therefore, all three have the same canonical conformation in their combining loops but differ in their scaffoldings. Eglin c (Leu45 at P1) binds to chymotrypsin much better than its Ala45 variant (the difference in standard free energy changes on binding is -5.00 kcal/mol). Similarly, turkey ovomucoid third domain (Leu18 at P1) binds to chymotrypsin much better than its Ala18 variant (the difference in standard free energy changes on binding is -4.70 kcal/mol). As these two differences are within the +/-400 cal/mol bandwidth (expected from the experimental error), one can conclude that the system is additive. On the basis that isoenergetic is isostructural, we expect that within both the P1 Ala pair and the P1 Leu pair, the conformation of the inhibitor's P1 side chain and of the enzyme's specificity pocket will be identical. This is confirmed, within the experimental error, by the available X-ray structures of complexes of bovine chymotrypsin Aalpha with eglin c () and with turkey ovomucoid third domain (). A comparison can also be made between the structures of P1 (Lys+)15 of bovine pancreatic trypsin inhibitor (Kunitz) ( and ) and of the P1 (Lys+)18 variant of turkey ovomucoid third domain (), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikingly different. Bovine pancreatic trypsin inhibitor with (Lys+)15 at P1 binds to chymotrypsin more strongly than its Ala15 variant (the difference in standard free energy changes on binding is -1.90 kcal/mol). In contrast, turkey ovomucoid third domain variant with (Lys+)18 at P1 binds to chymotrypsin less strongly than its Ala18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In this case, P1 Lys+ is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether the conformation of a P1 side chain in the complex matches that of an already determined one is at hand. Such a criterion may be useful in reducing the number of required X-ray crystallographic structure determinations. More importantly, the criterion can be applied to situations where direct determination of the structure is extremely difficult. Here, we apply it to determine the conformation of the Lys+ side chain in the transition state complex of a substrate with chymotrypsin. On the basis of kcat/KM measurements, the difference in free energies of activation for Suc-AAPX-pna when X is Lys+ and X is Ala is 1.29 kcal/mol. This is in good agreement with the corresponding difference for turkey ovomucoid third domain variants but in sharp contrast to the bovine pancreatic trypsin inhibitor (Kunitz) data. Therefore, we expect that in the transition state complex of this substrate with chymotrypsin, the P1 Lys+ side chain is deeply inserted into the enzyme's specificity pocket as it is in the (Lys+)18 turkey ovomucoid third domain complex with chymotrypsin.

Alanine↗
PubMed1999

Major subdomain rearrangement in HIV-1 reverse transcriptase simulated by molecular dynamics.

We have performed eight 1-ns in vacuo molecular dynamics simulations of HIV-1 reverse transcriptase (RT). Starting with the p66 thumb subdomain in an upright configuration, the p66 thumb moved down over the palm during six of the eight trajectories, in excellent agreement with the crystallographic structure of unliganded RT. The large rearrangement of the p66 thumb subdomain, its tip moving approximately 30 A, occurs during the first 30-200 ps. This approach may allow a detailed study of the processes involved in biologically significant conformational changes in macromolecules.

Crystallography, X-Ray↗
PubMed1999

Crystal structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA.

Human hnRNP A1 is a versatile single-stranded nucleic acid-binding protein that functions in various aspects of mRNA maturation and in telomere length regulation. The crystal structure of UP1, the amino-terminal domain of human hnRNP A1 containing two RNA-recognition motifs (RRMs), bound to a 12-nucleotide single-stranded telomeric DNA has been determined at 2.1 A resolution. The structure of the complex reveals the basis for sequence-specific recognition of the single-stranded overhangs of human telomeres by hnRNP A1. It also provides insights into the basis for high-affinity binding of hnRNP A1 to certain RNA sequences, and for nucleic acid binding and functional synergy between the RRMs. In the crystal structure, a UP1 dimer binds to two strands of DNA, and each strand contacts RRM1 of one monomer and RRM2 of the other. The two DNA strands are antiparallel, and regions of the protein flanking each RRM make important contacts with DNA. The extensive protein-protein interface seen in the crystal structure of the protein-DNA complex and the evolutionary conservation of the interface residues suggest the importance of specific protein-protein interactions for the sequence-specific recognition of single-stranded nucleic acids. Models for regular packaging of telomere 3' overhangs and for juxtaposition of alternative 5' splice sites are proposed.

Alternative Splicing↗
PubMed1999

Transvenous biventricular pacing for heart failure: can the obstacles be overcome?

Despite increasing evidence of hemodynamic benefit and long-term improvement in clinical status of congestive heart failure (CHF) patients with left ventricular and biventricular pacing, the risks and technical limitations of placing a permanent left ventricular pacing lead have prevented widespread clinical adoption of this therapy. Results of this and other recent investigations suggest it is necessary to target specific sites on the left ventricle to maximize hemodynamic benefit. However, limitations and variations of coronary vein anatomy, as well as patient safety, lead dislodgement, pacing thresholds, lead handling, and ease-of-use issues, present technical challenges for current transvenous permanent pacing lead designs. However, a new transvenous lead system based on an over-the-wire design appears to solve many of these problems and has proved feasible in acute clinical studies.

Animals↗
PubMed1999

A new approach to the effective preparation of plasma samples for rapid drug quantitation using on-line solid phase extraction mass spectrometry.

A procedure that permits rapid development of an optimized solid phase extraction (SPE) method for the analysis of drugs in plasma by on-line solid phase extraction-mass spectrometry (SPE-MS) has been developed. This procedure employs the concept of manipulating the pH and the percentage of organic solvent in the chromatographic mobile phase to affect the retention behaviors of both the matrix components and the analytes of interest. This resulted in the effective removal of matrix interferences from biological samples during SPE. During a the method development, only generic HPLC gradient approaches were needed, and multiple samples were pooled so that several SPE methods could be investigated at once. The analysis time per sample was 1.3 minutes. Thus, the time involved in the entire method development (analysis of a set of samples) was less than one hour. With the knowledge of the retention behaviors of the analytes with respect to the pH and the percentage of organic, it was then possible to compose an optimized SPE-MS method. This method consisted of a base/organic and then an acid/organic washing step, followed by a rapid gradient elution step. Due to the rigorous washing procedure, most matrix interferences were removed, and analytes eluted off the SPE sorbent suffered from very little matrix interference. Thus, quantitation of drugs in plasma by a single quadrupole mass spectrometer could be accomplished, something that was not possible when only a generic gradient was used for on-line SPE-MS. In addition, both external and internal calibration curves could be obtained for the concentration range from 5 to 500 ng/mL with correlation coefficients of 0.99 (using 1/x as a weighting factor) and relative standard deviations (RSDs) less than 10%. The results achieved were comparable to those obtained by the use of a triple quadrupole mass spectrometer. Moreover, the robustness of the method was tested by continuously injecting plasma samples. During 136 runs, the absolute peak area variation for these three basic drugs was less than 15% without taking the signal variation from the mass spectrometer into account. Significantly, the on-line developed method can be directly transferred to a 96-well format SPE plate.

Calibration↗
PubMed1999

Pivot residue: an analysis of domain motion in proteins.

In this study, we present an approach to identify some residues that represent the pivot points to experience conformational changes between open (unligand) and closed (ligand) forms of a protein. First, an angle, theta, formed by 4 consecutive Ca atoms in polypeptide backbones was introduced. The difference of this angle, deltatheta, from the equivalent residues between the open and the closed form was used to represent the local torsion changes in the protein structure, and the residue with the maximum among deltatheta was identified to be a pivot residue. We demonstrate the ability of our method by identifying the pivot residues from five proteins, Lysozyme mutates, Lactoferrin, Lay/Arg/Orn-binding protein, Calmodulin and Catabolit gene activator protein. These pivot residues are located at the hinges in the proteins, they are hinge points for the domain motion. These examples also show that the pivot residues are useful to distinguish the mechanism between shear motion and hinge motion in a protein.

Amino Acid Sequence↗
PubMed1999

Sequence pattern for the occurrence of N-glycosylation in proteins.

To further understand the occurrence of N-glycosylation, 21 nonhomologous proteins with Asn-x-Ser/Thr sequence were investigated. The results showed that some oligopeptides with Gly residues (G-x-y or y-x-G) are adjacent to the N-glycosylated sequences. These oligopeptides are not only essential for the structure and function of the proteins, but they are also found to be often proteolytic processing sites. These properties suggest that these oligopeptides may be a "sequence pattern" for the occurrence of N-glycosylation. The implications of the findings for protein structure and function are discussed.

Amino Acid Sequence↗
PubMed1999

Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices.

Most pockets in the human leukocyte antigen-group DR (HLA-DR) groove are shaped by clusters of polymorphic residues and, thus, have distinct chemical and size characteristics in different HLA-DR alleles. Each HLA-DR pocket can be characterized by "pocket profiles," a quantitative representation of the interaction of all natural amino acid residues with a given pocket. In this report we demonstrate that pocket profiles are nearly independent of the remaining HLA-DR cleft. A small database of profiles was sufficient to generate a large number of HLA-DR matrices, representing the majority of human HLA-DR peptide-binding specificity. These virtual matrices were incorporated in software (TEPITOPE) capable of predicting promiscuous HLA class II ligands. This software, in combination with DNA microarray technology, has provided a new tool for the generation of comprehensive databases of candidate promiscuous T-cell epitopes in human disease tissues. First, DNA microarrays are used to reveal genes that are specifically expressed or upregulated in disease tissues. Second, the prediction software enables the scanning of these genes for promiscuous HLA-DR binding sites. In an example, we demonstrate that starting from nearly 20,000 genes, a database of candidate colon cancer-specific and promiscuous T-cell epitopes could be fully populated within a matter of days. Our approach has implications for the development of epitope-based vaccines.

Alleles↗
PubMed1999

Intracytoplasmic sperm injection into zona-free human oocytes results in normal fertilization and blastocyst development.

Zona-free human oocytes are frequently encountered in in-vitro fertilization (IVF) laboratories. The oocytes escape out of the zona pellucida, following zona fracture, which can occur during oocyte retrieval or manipulation, but occasionally may be the result of increased zona fragility. Some of the zona-free oocytes are mature and morphologically healthy; nevertheless, all are typically discarded. In this report, we demonstrate that zona-free oocytes can be fertilized normally using intracytoplasmic sperm injection (ICSI) and can subsequently develop without zona to the blastocyst stage in vitro. We therefore suggest that those mature and morphologically normal zona-free oocytes may be rescued, fertilized with ICSI and then cultured to the blastocyst stage for subsequent transfer or cryopreservation.

Adult↗
PubMed1999

Intestinal cytokine response after gut ischemia: role of gut barrier failure.

OBJECTIVE: To investigate the effect of intestinal ischemia with and without a reperfusion injury on intestinal cytokine production and gut permeability. SUMMARY BACKGROUND DATA: In humans and in animal models, the gut has been implicated as a cytokine-producing organ after ischemia/reperfusion (I/R)-type injuries. Because of the limitations of in vivo models, it has been difficult to demonstrate directly that the gut releases cytokines after an I/R injury or whether there is a relation between the magnitude of the ischemic process and the cytokine response. METHODS: Ileal mucosal membranes from rats subjected to sham or 45 or 75 min of superior mesenteric occlusion (SMAO) or 45 minutes of SMAO and 30 minutes of reperfusion (SMAO 45/30) were mounted in the Ussing chamber system. Levels of tumor necrosis factor-alpha and interleukin-6 were serially measured in the mucosal and serosal reservoirs of the Ussing system, as was mucosal permeability as reflected by the passage of bacteria or phenol red across the ileal membrane. In a second group of experiments, Escherichia coli C25 was added to the mucosal reservoir to determine if the cytokine response would be increased. RESULTS: Mucosal and serosal levels of tumor necrosis factor-alpha were equally increased after SMAO, with the highest levels in the 75-minute SMAO group. The highest levels of interleukin-6 were found in rats subjected to 75 minutes of SMAO or SMAO 45/30; the serosal levels of interleukin-6 were four to sixfold higher than the mucosal levels. The addition of E. coli C25 resulted in a significant increase in the amount of interleukin-6 or tumor necrosis factor-alpha recovered from the mucosal reservoir. Increased ileal membrane permeability was observed only in rats subjected to 75 minutes of SMAO or SMAO 45/30. CONCLUSION: These results directly document that the levels of tumor necrosis factor-alpha and interleukin-6 released from the gut increase after an ischemic or I/R injury, such as SMAO, and that there is a relation between the magnitude of the gut ischemic or I/R insult and the cytokine response.

Animals↗
PubMed1999

Acoustic and current-pulse responses of identified neurons in the dorsal cochlear nucleus of unanesthetized, decerebrate gerbils.

In an effort to establish relationships between cell physiology and morphology in the dorsal cochlear nucleus (DCN), intracellular single-unit recording and marking experiments were conducted on decerebrate gerbils using horseradish peroxidase (HRP)- or neurobiotin-filled micropipettes. Intracellular responses to acoustic (tone and broadband noise bursts) and electric current-pulse stimuli were recorded and associated with cell morphology. Units were classified according to the response map scheme (type I to type V). Results from 19 identified neurons, including 13 fusiform cells, 2 giant cells, and 4 cartwheel cells, reveal correlations between cell morphology of these neurons and their acoustic responses. Most fusiform cells (8/13) are associated with type III unit response properties. A subset of fusiform cells was type I/III units (2), type III-i units (2), and a type IV-T unit. The giant cells were associated with type IV-i unit response properties. Cartwheel cells all had weak acoustic responses that were difficult to classify. Some measures of membrane properties also were correlated with cell morphology but to a lesser degree. Giant cells and all but one fusiform cell fired only simple action potentials (APs), whereas all cartwheel cells discharged complex APs. Giant and fusiform cells all had monotonic rate versus current level curves, whereas cartwheel cells had nonmonotonic curves. This implies that inhibitory acoustic responses, resulting in nonmonotonic rate versus sound level curves, are due to local inhibitory interactions rather than strictly to membrane properties. A complex-spiking fusiform cell with type III unit properties suggests that cartwheel cells are not the only complex-spiking cells in DCN. The diverse response properties of the DCN's fusiform cells suggests that they are very sensitive to the specific complement of excitatory and inhibitory inputs they receive.

Acoustic Stimulation↗
PubMed1999

Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors.

The migration of myogenic precursors to the vertebrate limb exemplifies a common problem in development - namely, how migratory cells that are committed to a specific lineage postpone terminal differentiation until they reach their destination. Here we show that in chicken embryos, expression of the Msx1 homeobox gene overlaps with Pax3 in migrating limb muscle precursors, which are committed myoblasts that do not express myogenic differentiation genes such as MyoD. We find that ectopic expression of Msx1 in the forelimb and somites of chicken embryos inhibits MyoD expression as well as muscle differentiation. Conversely, ectopic expression of Pax3 activates MyoD expression, while co-ectopic expression of Msx1 and Pax3 neutralizes their effects on MyoD. Moreover, we find that Msx1 represses and Pax3 activates MyoD regulatory elements in cell culture, while in combination, Msx1 and Pax3 oppose each other's trancriptional actions on MyoD. Finally, we show that the Msx1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3. Thus, we propose that Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors via direct protein-protein interaction. Our results implicate functional antagonism through competitive protein-protein interactions as a mechanism for regulating the differentiation state of migrating cells.

Animals↗
PubMed1999

Cytoplasmic maturation for activation of pig follicular oocytes cultured and arrested at metaphase I.

A large population (62-90%) of pig follicular oocytes can mature to metaphase II after culture for 48 h. However, a proportion (6-22%) remain in an immature stage at metaphase I (metaphase I-arrested). The main objective of this study was to determine whether the cytoplasm of metaphase I-arrested pig oocytes is capable of being activated by sperm penetration or parthenogenetic stimulation. After culture for 48 h, oocytes without a polar body (73% were shown to be at metaphase I after staining) and those with a polar body (94% were at metaphase II) were fertilized in vitro. A total of 69% and 62% of the oocytes were activated to form a female pronucleus, respectively, and the rate of polar body extrusion induced by fertilization in the activated oocytes was 90% (the first polar body) and 95% (the second polar body), respectively. When oocytes without and with a polar body were stimulated with an electric pulse, 53% and 81% of the oocytes were activated, respectively. The rate of polar body extrusion in the activated oocytes was 73% (the first polar body) and 79% (the second polar body), respectively. In contrast, young metaphase I oocytes cultured for 24 h had low (6%) or zero activation rate after in vitro fertilization or electric pulse stimulation. However, about one-third of the young metaphase I oocytes penetrated by spermatozoa after in vitro fertilization responded to electric pulse 12 h after insemination, and almost all (93%) were activated when they were stimulated 24 h after insemination. Patterns of polypeptide synthesis and histone H1 kinase activity were similar in metaphase I-arrested and metaphase II oocytes, and were characterized by increase in a 25 kDa polypeptide and by decrease in kinase activity. Although the first step of meiotic division is impaired, these results indicate that metaphase I-arrested oocytes are mature cytoplasmically.

Analysis of Variance↗