Biomedical subjects

G Shi

Publications and source records attributed to G Shi.

At least 19 recordsLinked to original sources

PubMed2006

Translational proteomics: developing a predictive capacity -- a review.

Over the past decade, proteomics has undergone a rapid development and radiation, diversifying across the biochemical landscape. While no single technique yet delivers complete proteomic coverage, application-specific adaptations afford significant opportunity for discovery and the development of predictive capacity (e.g. surrogate biomarker and clinical diagnostics). Targeted proteomic approaches, protein profiling strategies using affinity capture mass spectrometry and solution array represent realistic opportunities to deliver predictive capacity. The aim of this review is to provide an overview of proteomic technologies and how the outcomes delivered by such platforms may be translated into applications of predictive utility in clinical and basic science. In particular, recent applications in protein/peptide profiling (solid-phase affinity capture mass spectrometry and the targeted approach of antibody arrays) and the opportunities they afford researchers within the discipline of reproductive biology to develop new diagnostic and prognostic tests and surrogate biomarkers to improve the delivery of women's health care are considered.

Animals↗

PubMed2006

A novel NADP+-dependent L-1-amino-2-propanol dehydrogenase from Rhodococcus erythropolis MAK154: a promising enzyme for the production of double chiral aminoalcohols.

AIM: A novel NADP(+)-dependent L-1-amino-2-propanol dehydrogenase was isolated from Rhodococcus erythropolis MAK154, and characterized. METHODS AND RESULTS: The enzyme was inducibly produced on cultivation with aminoalcohols such as 1-amino-2-propanol, 1-amino-2-butanol and 2-aminocyclohexanol. The enzyme catalyses the NADP(+)-dependent oxidation of several aminoalcohols, and also the NADPH-dependent asymmetric reduction of an aminoketone compound to a double chiral aminoalcohol, d-pseudoephedrine. Amino acid sequence analysis showed that the enzyme might belong to the short-chain dehydrogenase/reductase family. CONCLUSIONS: NADP(+)-dependent L-1-amino-2-propanol dehydrogenase isolated from R. erythropolis MAK154 reversibly catalysed dehydrogenation of aminoalcohols, and exhibited a unique sterospecifity for the reduction reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The enzyme is a promising catalyst for the production of double chiral compound, d-pseudoephedrine, from prochiral substrate.

Alcohol Dehydrogenase↗

PubMed2005

Measuring EGJ opening patterns using high resolution intraluminal impedance.

The aim of this study was to adapt impedance methodology to study esophagogastric junction (EGJ) sphincter opening and compare opening patterns of the EGJ during deglutitive LES relaxation (dLESR) and transient LES relaxation (tLESR). We studied eight healthy subjects with a novel 12-lumen combined impedance/manometry catheter, the main element of which was a 6 cm sleeve sensor with six side hole sensors and six impedance rings spaced at 1 cm increments along its length. Subjects underwent an air infusion protocol after standard assessment and data tracings and isocontour plots were analysed to assess opening characteristics of the EGJ during dLESRs and tLESRs. Our results revealed that during dLESR the opening pattern was top to bottom, occurred in 0-2.7 s and in 29 of 35 (83%) cases the leading edge of the bolus was liquid. Opening during tLESR began between -7.8 and +8.6 s relative to the onset of nadir LES relaxation. The opening pattern during tLESR was bottom to top, occurred in 0-7.7 s, and in 22 of 29 (76%) the leading edge was liquid. These results support that impedance monitoring can be adapted to identify sphincter opening, to distinguish sphincter opening from sphincter relaxation, and to determine luminal contents during the opening period.

Adult↗

PubMed2004

Design and analysis of annular antenna arrays with different reflectors.

The design and performance of annular antenna arrays with reflectors is presented. Arrays with three shapes of reflectors are analysed and simulated. These include the corner reflector, the circular reflector and the elliptical reflector. Power-density distributions within the annular arrays with and without reflectors are obtained by using the FDTD method. Also, the image theory method is used to verify the FDTD results in one case. By comparing the power-density distribution pattern of all four of the array designs (three with different reflectors, one without reflector), it is readily seen in each case that the array with reflectors yields better power-efficiency than the array without reflectors and the elliptical reflector yields the best performance. Comparisons of each array are made using 4, 6 and 8 antennae in the annular array. By using the optimized results of the elliptical reflector, the requirement for the input-power level to each antenna is greatly reduced.

Computer Simulation↗

PubMed2003

Simultaneous quantification of seven active metabolites of roxifiban in human plasma by LC/MS/MS in the presence of an interfering displacer at millimolar concentrations.

Roxifiban (DMP 754) is a glycoprotein (GP) IIb/IIIa antagonist. Following oral administration to humans, roxifiban is metabolized to its primary active zwitterionic form, XV459, and several minor, active, hydrolyzed and hydroxylated metabolites, namely, M1a (DPC-AD3508), M1b (DPC-AD6128), M2 (SW156), M3 (DPC-AG2185), M8a (DPC-AF5814), and M8b (DPC-AF5818). Quantification of these metabolites in humans was not workable with a previous analytical method due to ion suppression of at least four of the analytes by a competitive displacer, DMP 728. This compound, which is another GP IIb/IIIa antagonist with very high affinity for the platelet receptor, was added to harvested blood samples in millimolar quantity to liberate XV459 from the GP IIb/IIIa receptor. An automated ion exchange solid phase extraction (IX-SPE) procedure was developed to selectively extract the seven metabolites of roxifiban and its deuterated internal standard while specifically excluding DMP 728. Among the six hydroxylation metabolites, there were two pairs of epimeric diastereomers (M1a/M1b and M8a/M8b) and one pair of geometric isomers (M2/M3), corresponding to three critical chromatographic pairs that needed to be base-line resolved because of the lack of specificity of MS/MS detection for these isomers. A new LC/MS/MS assay was developed to simultaneously quantify the seven metabolites in human plasma. The assay method was validated under GLP conditions over the concentration range of 0.5 to 80 nM for each of the analytes and successfully applied to assaying approximately 500 plasma samples from clinical trials.

Amidines↗

PubMed2003

The effect of glucagon-induced gastric relaxation on TLOSR frequency.

This study aimed to determine the effect of glucagon-induced gastric relaxation on the frequency of transient lower oesophageal sphincter relaxations (TLOSRs). Eight normal subjects (four male, age 18-52 y) were studied after a 6-h fast using a combined manometric barostat assembly. The recording was divided into two 1-h sessions: (1) a baseline period with the barostat set at minimal distending pressure (MDP) + 2 mmHg and (2) a period with continuous glucagon or placebo infusion with barostat set at MDP + 2 mmHg. Patients were studied on two different days and randomly received glucagon (4.8 microg kg(-1) bolus followed by 9.6 microg kg(-1) h(-1) infusion) on 1 day and placebo (saline) on another. Lower oesophageal sphincter (LOS) pressure, frequency of TLOSRs, and barostat bag volumes were determined for both placebo and glucagon infusion. Glucagon induced significant fundal relaxation compared with placebo (P < 0.05) and significantly decreased baseline LEOS pressure (P < 0.05). The frequency of TLOSRs was not altered by glucagon infusion compared with placebo. Despite causing substantial proximal stomach relaxation, glucagon did not increase TLOSR frequency. This suggests that the relevant gastric mechanoreceptors responsible for triggering TLOSRs do not respond to passive elongation.

Adolescent↗

PubMed2003

Reduced tLESR elicitation in response to gastric distension in fundoplication patients.

Transient lower esophageal sphincter relaxations (tLESRs) are vagally mediated in response to gastric cardiac distension. Nine volunteers, eight gastroesophageal reflux disease (GERD) patients, and eight fundoplication patients were studied. Manometry with an assembly that included a barostat bag was done for 1 h with and 1 h without barostat distension to 8 mmHg. Recordings were scored for tLESRs and barostat bag volume. Fundoplication patients had fewer tLESRs (0.4 +/- 0.3/h) than either normal subjects (2.4 +/- 0.5/h) or GERD patients (2.0 +/- 0.3/h). The tLESRs rate increased significantly in normal subjects (5.8 +/- 0.9/h) and GERD patients (5.4 +/- 0.8/h) during distension but not in the fundoplication group. All groups exhibited similar gastric accommodation (change in volume/change in pressure) in response to distension. Fundoplication patients exhibit a lower tLESR rate at rest and a marked attenuation of the response to gastric distension compared with either controls or GERD patients. Gastric accommodation was not impaired with fundoplication. This suggests that the receptive field for triggering tLESRs is contained within a wider field for elicitation of gastric receptive relaxation and that only the first is affected by fundoplication.

Adult↗

PubMed2002

The inherent cellular level of reactive oxygen species: one of the mechanisms determining apoptotic susceptibility of leukemic cells to arsenic trioxide.

Though reactive oxygen species (ROS) has been noticed to be involved in arsenic trioxide (As(2)O(3))-induced apoptosis of tumor cells, its role in apoptosis signaling remained to be elucidated. The objective of this work was to explore the association of the inherent cellular ROS level with the susceptibility of the tumor cells to apoptosis induction by As(2)O(3). Low concentration of As(2)O(3) was administered to cultured leukemic cell lines NB4, U937, HL60 and K562. The difference in apoptotic sensitivity was displayed among four cell types. ROS probes were incubated with the cells in the absence of As(2)O(3), and ROS was thus quantified relatively by flow cytometry. We manifested, in four cell types, the inherently existed difference in whole ROS quantity, and a positive correlation between the inherent ROS level and their apoptotic sensitivity to As(2)O(3). Furthermore, by interference using a ROS producer, we demonstrated that an elevation of ROS level would sensitize the cells to As(2)O(3)-induced apoptosis. The results of the present work suggested that the inherent ROS level might be determinative in tumor cells for their apoptotic susceptibility to As(2)O(3).

Antineoplastic Agents↗

PubMed2002

Distinct patterns of oesophageal shortening during primary peristalsis, secondary peristalsis and transient lower oesophageal sphincter relaxation.

This study characterized oesophageal shortening during secondary peristalsis and transient lower oesophageal sphincter relaxation (TLOSR) in an attempt to determine its contribution to the opening mechanism. Eight healthy subjects (four males, 26 +/- 1 years) had metal clips affixed at 0, +3, and +8 cm relative to the squamocolumnar junction (SCJ), defining two distal oesophageal segments. Axial clip movement was assessed with concurrent videofluoroscopy and manometry during primary peristalsis, secondary peristalsis and TLOSR. Clip-defined oesophageal segment length change was measured at 0.5-s intervals. The magnitude of the most distal segment shortening was least with TLOSR, greatest with primary peristalsis and intermediate with secondary peristalsis. Conversely, maximal overall oesophageal shortening during TLOSR, evidenced by SCJ movement, was similar to that during primary peristalsis. In 3/12 TLOSRs, the moment of LOS opening and gas reflux was optimally imaged; SCJ excursion was 0.3 +/- 0.1 cm prior to LOS opening and 1.4 +/- 0.7 cm immediately after gas reflux. The segmental pattern of oesophageal shortening was distinct during primary peristalsis, secondary peristalsis and TLOSR. During TLOSR, significant elevation of the SCJ occurred only after LOS opening, suggesting that this was a consequence of oesophageal distension induced by gas reflux rather than a component of the opening mechanism.

Adult↗

PubMed2001

A three-dimensional model of the human facilitative glucose transporter Glut1.

The human facilitative transporter Glut1 is the major glucose transporter present in all human cells, has a central role in metabolism, and is an archetype of the superfamily of major protein facilitators. Here we describe a three-dimensional structure of Glut1 based on helical packing schemes proposed for lactose permease and Glut1 and predictions of secondary structure, and refined using energy minimization, molecular dynamics simulations, and quality and environmental scores. The Ramachandran scores and the stereochemical quality of the structure obtained were as good as those for the known structures of the KcsA K(+) channel and aquaporin 1. We found two channels in Glut1. One of them traverses the structure completely, and is lined by many residues known to be solvent-accessible. Since it is delimited by the QLS motif and by several well conserved residues, it may serve as the substrate transport pathway. To validate our structure, we determined the distance between these channels and all the residues for which mutations are known. From the locations of sugar transporter signatures, motifs, and residues important to the transport function, we find that this Glut1 structure is consistent with mutagenesis and biochemical studies. It also accounts for functional deficits in seven pathogenic mutants.

Algorithms↗

PubMed2001

Unusual conformational changes in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by X-ray crystallography and NMR.

The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-A resolution with a crystallographic R factor of 0.191. The solution structure of HPPK in complex with Mg(2+) and beta,gamma-methyleneadenosine 5'-triphosphate (MgAMPPCP) has been determined using a simulated annealing protocol with 3,523 experimental NMR restraints. The root mean square deviation of the ensemble of 20 refined conformers that represent the solution structure from the mean coordinate set derived from them is 0.74 +/- 0.26 A for all backbone atoms and 0.49 +/- 0.22 A when residues Pro(14), Pro(44)-Gln(50), and Arg(84)-Pro(91) are excluded. Binding of MgADP causes significant changes in the conformation and dynamical property of three loops of HPPK that are involved in catalysis. A dramatic, unusual conformational change is that loop 3 moves away from the active center significantly with some residues moving by >17 A. The binding of MgADP also stabilizes loop 1 and loop 3 but makes loop 2 more mobile. Very similar conformational and dynamical changes are observed in the NMR solution structure of HPPK.MgAMPPCP. The conformational and dynamical changes may play important roles in both substrate binding and product release in the catalytic cycle.

Adenosine Diphosphate↗

PubMed2001

Identification of functional regions of Cbp3p, an enzyme-specific chaperone required for the assembly of ubiquinol-cytochrome c reductase in yeast mitochondria.

The Cbp3 protein of Saccharomyces cerevisiae is an enzyme-specific chaperone required for the assembly of ubiquinol-cytochrome c reductase of the mitochondrial respiratory chain. To gain preliminary insight into the role of Cbp3p during assembly, 29 independently isolated mutants were examined to define functional regions of the protein. Mutants were analyzed with respect to respiratory growth, ubiquinol-cytochrome c reductase assembly, and steady state amounts of enzyme subunits and Cbp3p. Three regions essential for Cbp3p activity were identified: regions 1 and 3 were required for Cbp3p function, while region 2 was necessary for protein stability. Mutation of Glu134 in region 1 (Cys124 through Ala140) impaired the ability of the Rieske FeS protein to assemble with the enzyme complex. Mutations targeted to region 3 (Gly223 through Asp229) primarily affected the 14 kDa subunit and cytochrome c(1) assembly. Gly223 was found especially sensitive to mutation and the introduction of charged residues at this site compromised Cbp3p functional activity. Region 2 (Leu167 through Pro175) overlapped the single hydrophobic domain of Cbp3p. Mutations within this area altered the association of Cbp3p with the mitochondrial membrane resulting in enhanced protein turnover. The role of the amino-terminus in Cbp3p activity was investigated using cbp3 deletion strains Delta12-23, Delta24-54, Delta56-96 and Delta12-96. All mutants were respiratory competent, indicating that residues 12-96 were not essential for Cbp3p function, stability or mitochondrial import. Analysis of carboxy-terminal deletion mutants demonstrated that the final 44 residues were not necessary for Cbp3p function; however, alterations in the secondary structure of the extreme carboxy-terminal 17 residues affected assembly protein activity.

Amino Acid Sequence↗

PubMed2001

Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: synthesis and biochemical and crystallographic studies.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. Like other enzymes in the folate pathway, HPPK is an ideal target for the development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from human and animals. Three bisubstrate analogues have been synthesized for HPPK and characterized by biochemical and X-ray crystallographic analyses. All three bisubstrate analogues consist of a pterin, an adenosine moiety, and a link composed of 2-4 phosphoryl groups. P(1)-(6-Hydroxymethylpterin)-P(2)-(5'-adenosyl)diphosphate (HP(2)A, 5) shows little affinity and inhibitory activity for E. coli HPPK. P(1)-(6-Hydroxymethylpterin)-P(3)-(5'-adenosyl)triphosphate (HP(3)A, 6) shows moderate affinity and inhibitory activity with K(d) = 4.25 microM in the presence of Mg(2+) and IC(50) = 1.27 microM. P(1)-(6-Hydroxymethylpterin)-P(4)-(5'-adenosyl)tetraphosphate (HP(4)A, 7) shows the highest affinity and inhibitory activity with K(d) = 0.47 microM in the presence of Mg(2+) and IC(50) = 0.44 microM. The affinity of MgHP(4)A for HPPK is approximately 116 and 76 times higher than that of MgADP and 6-hydroxymethylpterin, respectively. The crystal structure of HPPK in complex with 7 (HPPK.MgHP(4)A) has been determined at 1.85 A resolution with a crystallographic R factor of 0.185. The crystal structure shows that 7 occupies both HP- and ATP-binding sites and induces significant conformational changes in HPPK. The biochemical and structural studies of the bisubstrate analogues indicate that the bisubstrate analogue approach can produce more potent inhibitors for HPPK and the minimum length of the link for a bisubstrate analogue is approximately 7 A.

Adenosine Triphosphate↗

PubMed2001

Comparison of structural changes of cell surface carbohydrates in the eustachian tube epithelium of chinchillas infected with a Streptococcus pneumoniae neuraminidase-deficient mutant or its isogenic parent strain.

Six different lectin probes were used to examine alterations of the cell surface carbohydrates in the chinchilla eustachian tube (ET) lumen subsequent to the intranasal (i.n.) challenge with the Streptococcus pneumoniae parent strain, D39, or its isogenic derivative, DeltaNA1, which is deficient in neuraminidase NanA. The labelling pattern revealed that the binding of wheat germ agglutinin (WGA), Erythrina cristagalli lectin (ECL), peanut agglutinin (PNA), Bandeiraea simplicifolia lectin II (BSL II) and succinylated wheat germ agglutinin (SWGA) were increased in the lumenal surface of the ET in the D39 inoculated cohort compared to the uninfected control, which indicated that N-acetylglucosamine (GlcNAc) and D-galactose residues were exposed. Concurrently, decreased labelling with Sambucus nigra agglutinin (SNA) indicated that there were few sialic acid residues remaining in the ET epithelium subsequent to i.n. inoculation with D39. The DeltaNA1 neuraminidase deficient mutant, however, did not induce any significant changes in the lectin labelling patterns, and was comparable to that of the control cohort. We propose that products of the nanA gene have a significant impact on the changes of the carbohydrate moieties in the ET epithelium and may be responsible for the previously reported increased ability of the D39 parent to colonize the nasopharynx and invade the middle ear.

Animals↗

PubMed2001

Study of an Au colloid self-assembled electrode and its application to the determination of carbon monoxide.

A novel electrochemical sensor has been developed for the detection of carbon monoxide. The chemically modified electrode, prepared by reaction of cysteine and then an Au colloid of size approximately 15 nm with a platinum microelectrode, has excellent catalytic activity toward carbon monoxide, with an oxidation potential of +600 mV relative to the Ag/AgCl electrode. The CO gas sensor is based on an Au colloid self-assembled modified electrode as working electrode, an Ag/AgCl electrode as reference electrode, a Pt electrode as counter electrode, and a porous film which is in direct contact with the gas-containing atmosphere. The effects on the determination of CO of different internal electrolyte solutions of perchloric acid, hydrochloric acid, sulfuric acid, nitric acid, and phosphate buffer of different concentrations were also studied. The sensor is characterized by a short response time and highly reproducible detection of CO. This sensor can be used in the field of environmental monitoring and control.

Calibration↗

PubMed2001

Structure and dynamics of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

Folates are essential for life. Unlike mammals, most microorganisms must synthesize folates de novo. 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in folate pathway, and therefore, is an ideal target for developing novel antimicrobial agents. Because of its small size and high thermal stability, E. coli HPPK is also an excellent model enzyme for studying the mechanisms of enzymatic pyrophosphoryl transfer. We have determined the crystal structures of HPPK in the unligated form and in complex with HP, two Mg2+ ions, and AMPCPP (an ATP analog that inhibits the enzymatic reaction). Comparison of the two crystal structures reveals dramatic conformational changes of three flexible loops and many side chains and possible roles of the active site residues.

Adenosine Triphosphate↗

PubMed2001

Manometric heterogeneity in patients with idiopathic achalasia.

BACKGROUND & AIMS: In certain cases of achalasia, particularly those in early stages with minimal endoscopic or radiographic abnormalities, the diagnosis may rely on manometry, which is the most sensitive test for the disease. The aim of this study was to critically evaluate the manometric criteria in a population of patients with idiopathic achalasia. METHODS: Clinical histories and manometric recordings of 58 patients with idiopathic achalasia and 43 control subjects were analyzed with regard to esophageal body contraction amplitude, peristaltic effectiveness in terms of both completeness and propagation velocity, lower esophageal sphincter (LES) resting pressure, LES relaxation pressure, and intraesophageal-intragastric pressure gradient. Variants of achalasia were defined by finding manometric features that significantly differed from the remainder of achalasia patients, such that the diagnosis might be questioned. RESULTS: Four manometrically distinct variants were identified. These variants were characterized by (1) the presence of high amplitude esophageal body contractions, (2) a short segment of esophageal body aperistalsis, (3) retained complete deglutitive LES relaxation, and (4) intact transient LES relaxation. In each instance, the most extreme variant is discussed and compared with the remainder of the achalasia population and with controls. CONCLUSIONS: The significance in defining these variants of achalasia lies in the recognition that these sometimes confusing manometric findings are consistent with achalasia when combined with additional clinical data supportive of the diagnosis. Furthermore, such variants provide important clues into the pathophysiology of this rare disorder.

Adult↗

PubMed2001

Mercurial sensitivity of aquaporin 1 endofacial loop B residues.

The water channel protein aquaporin-1 (AQP1) has two asparagine-proline-alanine (NPA) repeats on loops B and E. From recent structural information, these loops are on opposite sides of the membrane and meet to form a pore. We replaced the mercury-sensitive residue cysteine 189 in AQP1 by serine to obtain a mercury-insensitive template (C189S). Subsequently, we substituted three consecutive cysteines for residues 71-73 near the first NPA repeat (76-78) in intracellular loop B, and investigated whether they were accessible to extracellular mercurials. AQP1 and its mutants were expressed in Xenopus laevis oocytes, and the osmotic permeability (P(f)) of the oocytes was determined. C189S had wild-type P(f) but was not sensitive to HgCl(2). Expression of all three C189S cysteine mutants resulted in increased P(f), and all three mutants regained mercurial sensitivity. These results, especially the inhibitions by the large mercurial p-chloromercunbenzene-sulfonic acid (pCMBS) ( approximately 6A wide), suggest that residues 71-73 at the pore are accessible to extracellular mercurials. A 30-ps molecular dynamics simulation (at 300 K) starting with crystallographic coordinates of AQP1 showed that the width of the pore bottleneck (between Connolly surfaces) can vary (w(avg) = 3.9 A, sigma = 0.75; hydrated AQP1). Thus, although the pore width would be > or = 6 A only for 0.0026 of the time, this might suffice for pCMBS to reach residues 71-73. Alternative explanations such as passage of pCMBS across the AQP1 tetramer center or other unspecified transmembrane pathways cannot be excluded.

Amino Acid Motifs↗