Biomedical subjects

X Hu

Publications and source records attributed to X Hu.

At least 181 records · Page 10Linked to original sources

PubMed1999

Voltammetric response and determination of DNA with a silver electrode.

A current from DNA was obtained using a silver electrode with low overpotentials for the first time. Experimental results revealed that the voltammetric response of DNA was attributed to the redox reactions of purine bases. It was also shown that such a method provided a convenient and practical way to determine DNA. A linear dependence of the peak currents on ssDNA concentrations was observed in the range 0.5-2.5 microg/mL. The relative standard deviation was 3.5% for six successive determinations at 0.5 microg/mL. The detection limit was 50 ng/mL. Influence of the structure and the length of the nucleic acids on their electrochemical behavior was discussed. In view of the merits of the silver electrode, this technique might provide new possibilities for further electrochemical research and determination of nucleic acids.

Adenine↗

PubMed1999

Differential catalytic efficiency of allelic variants of human glutathione S-transferase Pi in catalyzing the glutathione conjugation of thiotepa.

Alkylating agents are extensively used in the treatment of cancer. The clinical usefulness of this class of anticancer drugs, however, is often limited by the emergence of drug-resistant tumor cells. Increased glutathione (GSH) conjugation through catalysis by GSH S-transferases (GSTs) is believed to be an important mechanism in tumor cell resistance to alkylating agents. In the present study, we report that the allelic variants of human Pi class GST (hGSTP1-1), which differ in their primary structures at amino acids in positions 104 and/or 113, exhibit significant differences in their activity in the GSH conjugation of alkylating anticancer drug thiotepa. Mass spectrometry revealed that the major product of the reaction between thiotepa and GSH was the monoglutathionyl-thiotepa conjugate. While nonenzymatic formation of monoglutathionyl-thiotepa was negligible, the formation of this conjugate was increased significantly in the presence of hGSTP1-1 protein. The hGSTP1-1-catalyzed GSH conjugation of thiotepa was time and protein dependent and followed Michaelis-Menten kinetics. The catalytic efficiency of hGSTP1-1(I104, A113) variant was approximately 1.9- and 2.6-fold higher compared with hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113) isoforms, respectively. The results of the present study indicate that the hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to thiotepa, and that subjects homozygous for the hGSTP1-1(I104,A113) allele, which is most frequent in human populations, are likely to be at a greater risk for developing GST-mediated resistance to thiotepa than heterozygotes or homozygotes with valine 104 background.

Alleles↗

PubMed1999

Exchange of conductance and gating properties between gap junction hemichannels.

Gap junction channels span the membranes of two adjacent cells and allow the gated transit of molecules as large as second messengers from cell to cell. The structure of the gap junction channel pore is not resolved. For identification of pore determinants we used a chimera of two connexins, cx46 and cx32E(1)43, that form membrane channels with distinct unit conductances and channel kinetics. Exchange of the first transmembrane segment (M1) between these connexins resulted in a chimera that exhibited most of the channel properties of the M1 donor, including single channel conductance, channel kinetics, and the preference to dwell at a subconductance level. The M1 segment thus appears to be an important determinant of conductance and gating properties of connexin channels.

Animals↗

PubMed1999

Promoter activity of mouse kappa opioid receptor gene in transgenic mouse.

The biological activity of mouse kappa opioid receptor (KOR) gene promoter was examined in transgenic mice using a beta-galactosidase (lacZ) reporter strategy for the first time. A lacZ cDNA was inserted at the 5th amino acid in the coding region of a mouse KOR genomic segment containing 3 kb of the 5' regulatory region, to generate a Kor-lacZ fusion gene which was then used to generate transgenic mice. The expression of transgene was demonstrated at the RNA level by reverse transcription-polymerase chain reaction (RT-PCR), and at the protein level by in situ lacZ enzyme assay. From studying three independent transgenic mouse lines that express this transgene, it is concluded that Kor-lacZ expression begins at embryonic day 9.5 (E9.5) and increases in several brain areas and neural tube as embryos develop. At E12.5 and E13.5, Kor-lacZ expression is found primarily in the mantle layer of midbrain, hindbrain and medulla oblongata, cranial ganglion and vagus nerve. At E15.5 and E17.5, the transgene is expressed in eye, ear, neopallial cortex, caudate putamen, lateral ventricle, thalamus, hypothalamus and pons. Therefore, the 3 kb 5' regulatory sequence of the mouse KOR gene is functional in transgenic animals and directs a specific expression pattern recapitulating that of the endogenous KOR gene expression during developmental stages. However, in adult animals, this transgene is only expressed in the brain, indicating that the regulatory information for peripheral expression in the adult is not encoded within this 3 kb upstream sequence.

Age Factors↗

PubMed1999

Differential protection against benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced DNA damage in HepG2 cells stably transfected with allelic variants of pi class human glutathione S-transferase.

The pi class glutathione S-transferase (GSTP1-1), which is polymorphic in human populations, is believed to play an important role in detoxification of the ultimate carcinogen of widespread environmental pollutant benzo[a]pyrene [(+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide [(+)-anti-BPDE]]. The allelic variants of human GSTP1-1 (hGSTP1-1) differ in their structures by the amino acids in positions 104 (isoleucine or valine) and/or 113 (valine or alanine). Here, we have determined the protective effect of overexpression of allelic variants of hGSTP1-1, through stable transfection in HepG2 cells, against (+)-anti-BPDE-induced DNA modification. Clonal transfectants of HepG2 cells corresponding to the three allelic variants of hGSTP1-1 [(I104,A113), (V104,A113), and (V104,V113), denoted hGSTP1(IA), hGSTP1(VA), and hGSTP1(VV), respectively] with similar levels of hGSTP1 protein were identified and characterized for their GST activity and (+)-anti-BPDE-induced DNA modification. The glutathione S-transferase activity toward (+)-anti-BPDE was significantly higher (approximately 3.0-3.6-fold) in cells transfected with hGSTP1(VA) [HepG2(VA)] and hGSTP1(VV) [HepG2(VV)] compared with hGSTP1(IA) transfectant [HepG2(IA)]. The formation of (+)-anti-BPDE-DNA adducts was significantly reduced in HepG2(VA) and HepG2(VV) cells compared with cells transfected with insert-free vector (HepG2-vect). Maximum protection against (+)-anti-BPDE-induced DNA damage was afforded by the hGSTP1(VV) isoform. The results of this study indicate that the allelic variants of hGSTP1-1 significantly differ in their ability to provide protection against (+)-anti-BPDE-induced DNA damage. Thus, hGSTP1-1 polymorphism may be an important factor in differential susceptibility of individuals to tumorigenesis induced by benzo[a]pyrene.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1999

The mixed lineage kinase DLK utilizes MKK7 and not MKK4 as substrate.

Mixed lineage kinases DLK (dual leucine zipper-bearing kinase) and MLK3 have been proposed to function as mitogen-activated protein kinase kinase kinases in pathways leading to stress-activated protein kinase/c-Jun NH2-terminal kinase activation. Differences in primary protein structure place these MLK (mixed lineage kinase) enzymes in separate subfamilies and suggest that they perform distinct functional roles. Both DLK and MLK3 associated with, phosphorylated, and activated MKK7 in vitro. Unlike MLK3, however, DLK did not phosphorylate or activate recombinant MKK4 in vitro. In confirmatory experiments performed in vivo, DLK both associated with and activated MKK7. The relative localization of endogenous DLK, MLK3, MKK4, and MKK7 was determined in cells of the nervous system. Distinct from MLK3, which was identified in non-neuronal cells, DLK and MKK7 were detected predominantly in neurons in sections of adult rat cortex by immunocytochemistry. Subcellular fractionation experiments of cerebral cortex identified DLK and MKK7 in similar nuclear and extranuclear subcellular compartments. Concordant with biochemical experiments, however, MKK4 occupied compartments distinct from that of DLK and MKK7. That DLK and MKK7 occupied subcellular compartments distinct from MKK4 was confirmed by immunocytochemistry in primary neuronal culture. The dissimilar cellular specificity of DLK and MLK3 and the specific substrate utilization and subcellular compartmentation of DLK suggest that specific mixed lineage kinases participate in unique signal transduction events.

Animals↗

PubMed1999

New light on allostery: dynamic resonance Raman spectroscopy of hemoglobin kempsey.

On the basis of static and time-resolved resonance Raman spectroscopy of HbA and of a mutant, HbK (Dalpha99N), a specific reaction coordinate is proposed for the allosteric transition in human hemoglobin. The heme is held between proximal (F) and distal (E) helices, whose orientation is responsive to forces generated by ligation and deligation. The E and F helices are in turn tethered via H-bonds to the A and H helices. These outer helices follow the E-F motion, thereby repositioning the N- and C-termini, which form the intersubunit salt bridges in the T quaternary structure. When the T state interface is weakened by Asp --> Asn substitution at a quaternary H-bond (HbK), the Fe-His bond is relaxed and becomes responsive to allosteric effectors. The same E-F motion is observed in HbK, but the A-H following motion is delayed, relative to HbA, as is the Asn H-bond formation.

Allosteric Regulation↗

PubMed1999

Mapping of residues in the NADP(H)-binding site of proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. A study of structure and function.

Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical betaalphabetaalphabeta dinucleotide binding fold is responsible for binding NADP(H). A model defining betaA, alphaB, betaB, betaD, and betaE of this domain is presented. To test this model, four single cysteine mutants (cfbetaA348C, cfbetaA390C, cfbetaK424C, and cfbetaR425C) were introduced into a functional cysteine-free transhydrogenase. Also, five cysteine mutants were constructed in the isolated domain III of Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic characterizations, effects of sulfhydryl-specific labeling with N-ethylmaleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined. The results are consistent with the view that, in agreement with the model, beta-Ala348, beta-Arg350, beta-Ala390, beta-Asp392, and beta-Lys424 are located in or close to the NADP(H) site. More specifically, beta-Ala348 succeeds betaB. The remarkable reactivity of betaR350C toward NNADP suggests that this residue is close to the nicotinamide moiety of NADP(H). beta-Ala390 and beta-Asp392 terminate or succeed betaD, and are thus, together with the region following betaA, creating the switch point crevice where NADP(H) binds. beta-Asp392 is particularly important for the substrate affinity, but it could also have a more complex role in the coupling mechanism for transhydrogenase.

Amino Acid Sequence↗

PubMed1999

Gender related differences in ATP-dependent transport of dinitrophenyl-glutathione conjugate across murine canalicular liver plasma membrane.

The present study reports gender related differences in ATP-dependent transport of dinitrophenyl-glutathione (GSH) conjugate (DNP-SG), a model GSH xenobiotic conjugate, across murine canalicular liver plasma membrane (cLPM). ATP-dependent transport of DNP-SG across female A/J mouse cLPM was mediated by two components, a high-affinity and a low-affinity component, with corresponding Km of 18 microM (Vmax 0.02 nmol/min.mg) and 500 microM (Vmax 0.23 nmol/min.mg), respectively. On the other hand, only one component for the ATP-dependent transport of DNP-SG was observed in male mouse cLPM (K(m) 130 microM; Vmax 0.18 nmol/min.mg). Moreover, the rate of ATP-dependent transport of DNP-SG was markedly higher in the cLPM fraction of male mouse compared with that of the female. Presence of two transport components in female mouse cLPM, but only one system in the cLPM fraction of male mouse, was confirmed by measuring DNP-SG mediated stimulation of ATP hydrolysis (DNP-SG ATPase activity). To the best of our knowledge, the present study is the first report on gender related differences in ATP-dependent murine canalicular transport of GSH conjugates.

Adenosine Triphosphate↗

PubMed1999

Site-directed mutagenesis of charged and potentially proton-carrying residues in the beta subunit of the proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. Characterization of the beta H91, beta D392, and beta K424 mutants.

Conserved and semiconserved acidic and basic residues of the beta subunit of the proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli potentially involved in proton pumping were investigated. Out of 16 charged residues studied, 6 have not been previously investigated. The most dramatic effects of mutation were observed with beta H91, beta D392, and beta K424. beta H91E showed a pronounced shift of the pH optimum for both reduction of thio-NADP+ by NADH (forward reaction) and reduction of 3-acetylpyridine-NAD+ by NADPH (reverse reaction) to lower pH. This mutant catalyzed a cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADP(H) with a pH profile also shifted toward a lower pH. These results are consistent with a mechanism where the normal forward and reverse reactions are indeed limited by protonation/deprotonation of beta H91. The cyclic reaction was affected by mutations of beta H91, probably through conformational changes involving the active NADP(H) site. The beta D392A mutant was inactive with regard to forward and reverse reactions, but showed a wild-type-like pH dependence for the partly active cyclic reaction. However, Km,app for NADP(H) in this reaction was elevated 50-100-fold, suggesting that beta D392 is located in or near the NADP(H)-binding site. Transhydrogenases contain a conserved beta K424-beta R425-beta S426 sequence that has been proposed to be important for NADP(H) binding. beta K424R was strongly inhibited and showed an 18-fold increased Km,app for NADPH in the reverse reaction as compared to wild type. Consequently, this mutation affected all NADP(H)-linked activities and essentially abolished the unspecific interaction of NAD(H) with this site. The pH dependences of the forward and reverse reactions, as well as the cyclic reaction, were shifted to a lower pH as compared to the wild-type enzyme, and the salt dependence was also altered.

Aspartic Acid↗

PubMed1999

Optical mapping of DNA polymerase I action and products.

Single molecule approaches to the characterization of biochemical systems offer an intrinsically simple and direct approach to address difficult, previously unyielding problems. Optically based approaches have recently been used to construct high resolution, ordered restriction maps from a variety of clone types. Advancements in surface technologies have enabled the reliable elongation and fixation of large DNA molecules onto specially derivatized substrates with retention of biochemical accessibility. In this study, the addition of fluorescently labeled nucleotides to surface-mounted DNA molecules by the action of DNA polymerase I is investigated using fluorescence microscopy to image individual template molecules. Molecules undergoing nick translation and containing only a few fluorochromes are readily imaged. These novel results suggest that surface-bound molecules may serve as a substrate for a broad range of enzymatic actions, and may offer new routes to analysis when coupled to advanced imaging techniques.

Bacteriophage lambda↗

PubMed1999

Applications of high-resolution echoplanar spectroscopic imaging for structural imaging.

Echoplanar spectroscopic imaging (EPSI) was introduced as a fast alternative for spectroscopic imaging and has been recently implemented on clinical scanners. With further advances in gradient hardware and processing strategies, EPSI can be used to obtain spectroscopic images whose spatial resolution parallels that of conventional anatomic images within clinically acceptable acquisition time. The present work demonstrates that high-resolution EPSI can be used to derive structural images for applications in which spectroscopic information is beneficial. These applications are chemical shift (fat-water) imaging, narrow bandwidth imaging, and T2* mapping. In this paper, the EPSI sequence design and processing strategies are detailed and experimental results in normal volunteers are presented to illustrate the potential of using EPSI in imaging anatomic structures.

Adipose Tissue↗

PubMed1999

Further evaluation of the initial negative response in functional magnetic resonance imaging.

The initial negative response (i.e., the dip) in the functional MR signal at stimulus onset has aroused much interest. Such a response is consistent with optical imaging data and can be potentially mapped to generate spatially more specific maps. However, there are still controversies regarding the exact origin of the initial response. In particular, experimental reports of its echo-time dependence have been inconsistent. Furthermore, several investigators have suggested the possibility of an apparent dip that may arise artifactually when the interstimulus interval (ISI) is not sufficiently long. The present study investigates the echo-time dependence of the initial response and the effect of the ISI on the initial response. Experimental results obtained at TEs of 21, 30, and 45 msec demonstrate that the initial dip has a TE dependence that is in agreement with a T2* contrast, and thereby consistent with a blood oxygenation level-dependent origin. At an ISI of 90 sec, a statistically significant initial negative response was detected and shown to be indistinguishable from that observed at an ISI of 45 sec, which was used in our previous studies, indicating that the initial negative response observed at 4 T is not a consequence of short ISI.

Echo-Planar Imaging↗

PubMed1999

Analysis of functional magnetic resonance imaging data using self-organizing mapping with spatial connectivity.

Commonly used methods in analyzing functional magnetic resonance imaging (fMRI) data, such as the Student's t-test and cross-correlation analysis, are model-based approaches. Although these methods are easy to implement and are effective in analyzing data obtained with simple paradigms, they are not applicable in situations in which patterns of neuronal response are complicated and when fMRI response is unknown. In this work, Kohonen's self-organizing mapping (SOM), which is a model-free approach, is adapted for analyzing fMRI data. Because spatial connectivity is an important function in the identification of activation sites in functional brain imaging, it is incorporated into the SOM algorithm. Receiver operating characteristic analysis on simulated data shows that the new algorithm achieves measurable improvement over the standard algorithm. The applicability of the new algorithm is demonstrated on experimental data.

Algorithms↗

PubMed1999

Detection of the early negative response in fMRI at 1.5 Tesla.

Recent experimental studies have revealed an initial decrease in magnetic resonance (MR) signal that is consistent with optical imaging results. This initial response, thought to arise from a transient increase in deoxyhemoglobin concentration, is probably more localized to the site of neuronal activation. However, with MR imaging, this early response has only been demonstrated at high fields. In this paper, the observation of the initial response at 1.5 T is reported. Compared with results obtained at 4 T, the present study reveals that the initial response grows more rapidly with the field strength than the late positive response, suggesting a more microvascular origin of the initial response.

Adult↗

PubMed1999

A new view of Starling's hypothesis at the microstructural level.

In this paper we quantitatively investigate the hypothesis proposed by Michel (Exp. Physiol. 82, 1-30, 1997) and Weinbaum (Ann. Biomed. Eng. 26, 1-17, 1998) that the Starling forces are determined by the local difference in the hydrostatic and colloid osmotic pressure across the endothelial surface glycocalyx, which we propose is the primary molecular sieve for plasma proteins, rather than the global difference in the hydrostatic and oncotic pressure across the capillary wall between the plasma and tissue, as has been universally assumed until now. A spatially heterogeneous microstructural model is developed to explain at the cellular level why there is oncotic absorption at low capillary pressures in the short-lived transient experiments of Michel and Phillips (J. Physiol. 388, 421-435, 1987) on frog mesentery capillary, but a small positive filtration once a steady state is achieved. The new model also predicts that the local protein concentration behind the surface glycocalyx can differ greatly from the tissue protein concentration, since the convective flux of proteins through the orifice-like pores in the junction strand will greatly impede the back diffusion of the proteins into the lumen side of the cleft when the local Peclet number at the orifice is >1. The net result is that the filtration in the capillaries is far less than heretofore realized and there may be no need for venous reabsorption.

Animals↗

PubMed1999

Quality control of postoperative acute pain service.

To establish an effective method of continuous quality control of acute pain service, a retrospective study on incident reporting during postoperative analgesia period was conducted. Incidents were reported and analyzed in 1507 patients who received epidural postoperative analgesia, and the results of satisfaction of pain relief was compared with those of incident analysis. In this study, an incident was defined as any factor that might or had affected patient's safety during analgesia period. Our results showed that 1203 incidents were reported in 641 of 1507 patients, of which 122 incidents were critical. 78.3% of all incidents were detected by acute pain service stuff. The most common incidents included complications, insufficient analgesia and problems with delivery circuits. Human factors were involved in 28.9% of the incidents, most being associated with technical failure due to unskillfulness, poor communications between APS stuff and patients and lack of cooperation with surgeons and nurses. The general satisfaction rate of the patients was 90.8%. There was a very significant difference between the satisfaction of the patients who suffered from incidents and who did not (P < 0.001). It is concluded that incidents affect the satisfaction of the patients who received postoperative pain relief. Incident reporting is a more effective method for quality control of acute pain service.

Analgesia, Epidural↗

PubMed1999

Two-body in vitro wear study of some current dental composites and amalgams.

STATEMENT OF PROBLEM: Wear resistance of restorative materials is a major concern in clinical practice for restorations involving occlusion. The relatively poor wear resistance of dental composites in stress-bearing posterior situations has restricted even wider clinical application of this type of restorative material. PURPOSE: This in vitro study compared the relative wear resistance of a selection of current dental composites and amalgams under cyclic loading to explore the wear mechanisms operating on these materials and to assess their relative potential clinical wear resistance under variable masticatory loads. MATERIAL AND METHODS: A 2-body in vitro wear test was undertaken on a selection of 2 Ultrafine Compact-Filled composites, 1 microfilled composite, and 3 dental amalgams. An alternating sine curve load (0.5 to 15 N) was applied to the wear surface through a rotating countersample with a purpose-built system to simulate the variable loading pattern that occurs during mastication. RESULTS: Under conditions simulating the repetitive cyclic loading pattern that may occur intraorally in high stress occlusal contact situations, the Ultrafine Compact-Filled composites exhibited a delamination wear pattern and had a significantly lower wear resistance than the amalgams. By comparison, the microfilled composite (Silux Plus) displayed an improved wear resistance that was superior to some amalgams. Dispersalloy had the best wear resistance among the materials selected. CONCLUSION: The wear of Ultrafine Compact-Filled composite and microfilled composite differed and reflect different operative wear mechanisms. For amalgams, the size, shape, and composition of the particles had an effect on the wear resistance of the materials.

Analysis of Variance↗