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Biomedical subjects

Q Xue

Publications and source records attributed to Q Xue.

At least 73 records · Page 4Linked to original sources
PubMed1996

Trichodiene synthase. Probing the role of the highly conserved aspartate-rich region by site-directed mutagenesis.

Trichodiene synthase catalyzes the cyclization of farnesyl diphosphate to the sesquiterpene hydrocarbon trichodiene. The enzyme normally requires a divalent cation, Mg2+, which can be substituted by Mn2+. Trichodiene synthase from Fusarium sporotrichioides has a highly conserved aspartate rich region, aa 100-104 (DDSKD). Three mutants were constructed by site-directed mutagenesis in which each aspartate residue was individually replaced by glutamate. The mutants were each overexpressed and purified to homogeneity. The importance of Asp100 and Asp101 for catalysis was established by the observation of an increase in Km as well as a reduction in kcat in the corresponding Glu mutants. Replacement of the Asp104 residue with Glu had little effect on either Km or kcat. All three mutants produced anomalous sesquiterpene products in addition to trichodiene when incubated with farnesyl diphosphate. Interestingly, when Mg2+ was replaced by Mn2+ in the incubation buffer, the kcat/Km of both wild type trichodiene synthase and the D104E dropped significantly, while those of the other two mutants were not much affected. The proportion of anomalous products increased significantly when the D100E and D101E mutants were incubated in the presence of Mn2+. These observations all lend weight to the proposal that the aspartate residues mediate substrate binding by chelation of the divalent metal ion. Asp100 and Asp101 appear to play a relatively more important role than Asp104.

Aspartic Acid↗
PubMed1996

Establishment of a metastatic model of human hepatocellular carcinoma in nude mice via orthotopic implantation of histologically intact tissues.

A highly metastatic LCI-D20 model of human hepatocellular carcinoma (HCC) was obtained via orthotopic implantation of a histologically preserved metastatic tumour selected from 1 of 30 surgical specimens, and had been maintained for 18 passages in nude mice. All mice with transplanted tumours exhibited 100% transplantability and metastatic ability, as well as various manifestations reminiscent of tumour behaviour in HCC patients. These included: local growth, regional invasion, spontaneous metastasis to liver, lymph nodes and lungs, and peritoneal seeding. Histological characteristics of the LCI-D20 tumour were similar to those of the original tumour. Karyotypic analysis revealed heteroploid cells. Immunohistochemically, expression of AFP and HBxAG was shown. Our nude mouse model with its high metastatic rate and short latency period could be an interesting tool for the study of human HCC.

Animals↗
PubMed1996

Determination of lactate dehydrogenase isoenzymes in single lymphocytes from normal and leukemia cell lines.

This work demonstrates that our previously developed technique for single-erythrocyte analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) can be applied to study individual lymphocytes, with some modification in the cell lysing procedure. A tesla coil was shown to be capable of lysing the lymphocyte cells inside the capillary. The electromagnetic field induced by the tesla coil was believed to be responsible for breaking the cell membrane. The lactate dehydrogenase (LDH) isoenzyme activities and relative ratios between different LDH isoenzymes were measured for normal lymphocytes as well as B-type and T-type acute lymphoblastic leukemia cells. Both the LDH activity and the isoenzyme ratios show large variations among individual cells. The former is expected due to variations in cell size. The latter implies that single-cell measurements are less useful than the average values over a cell population as markers for leukemia.

Biomarkers, Tumor↗
PubMed1996

Metastatic models of human liver cancer in nude mice orthotopically constructed by using histologically intact patient specimens.

In this study of orthotopic implantation of histologically intact surgical specimens, the authors constructed metastatic models of human hepatocellular carcinoma (HCC) in nude mice. Histologically intact human liver cancer specimens, derived from patients, were implanted directly into the liver of nude mice, and their orthotopic growth and metastases were observed. The transplantability and metastatic rate of two specimen groups (primary and metastatic lesions) were analysed. alpha-Fetoprotein (AFP) was also determined in transplanted tumours by an immunohistochemical method. Orthotopic growth was observed in 14 of 30 transplanted specimens and formation of metastases in 7 cases, which exhibited the variety of clinical behaviours seen in patients with HCC. These behaviours included local growth, regional invasion, spontaneous intrahepatic, lymph node and lung metastasis and peritoneal seeding. In two groups the growth rate of metastatic lesions following implantation was clearly higher than that of primary tumours. Chromosome analysis from locally growing tumours confirmed their morphologically human origin. An immunohistochemical study showed that implanted tumours originating from AFP-positive specimens maintained AFP expression. These results indicated that the animal models should prove valuable for developing new treatment modalities and studying the mechanism of metastasis of human HCC.

Animals↗
PubMed1996

Analysis of interval measurements on CSE multilead reference ECGs.

As part of the International Electrotechnical Commission's (IEC) efforts to develop standards for interpretive electrocardiographs, electrocardiograms (ECGs) of the Common Standards of Electrocardiography (CSE) multilead measurement database were processed to (1) determine whether original ECG data would offer any advantage over cycle-repeated (artificial) ECGs and (2) compare the "clean" ECGs selected for interval measurement compliance testing (n = 100) with the remaining 25 "noisy" or nonsinus rhythm ECGs. Two sets of CSE measurement ECGs, namely, 125 original ECGs of the MO1 series and 125 artificial ECGs of the MA1 series were divided into 100 IEC-selected clean ECGs with good P and T waves and 25 noisy or nonsinus ECGs and processed for global wave duration and interval measurements (P duration, PR interval, QRS duration, and QT interval). The measured duration and interval values were compared against the CSE reference values (medians of referee values for 25 physician overread ECGs and medians of interpretive programs for the rest) to compute the measurement "differences". Also, the data of the median cardiac complex were evaluated for noise content. The original ECG data gave consistently smaller differences for all of the four measurements than the differences with the artificial ECG data. The noise levels in the median complexes formed from the original ECG data were significantly lower than the noise levels in the median complexes from the artificial ECG data. The noise levels in the medians of the 100 clean ECGs were lower than the noise levels in the medians of the 25 noisy ECGs for both the original and artificial ECG data.

Algorithms↗
PubMed1996

Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant.

We introduced the potato proteinase inhibitor II (PINII) gene (pin2) into several Japonica rice varieties, and regenerated a large number of transgenic rice plants. Wound-inducible expression of the pin2 gene driven by its own promoter, together with the first intron of the rice actin 1 gene (act1), resulted in high-level accumulation of the PINII protein in the transgenic plants. The introduced pin2 gene was stably inherited in the second, third, and fourth generations, as shown by molecular analyses. Based on data from the molecular analyses, several homozygous transgenic lines were obtained. Bioassay for insect resistance with the fifth-generation transgenic rice plants showed that transgenic rice plants had increased resistance to a major rice insect pest, pink stem borer (Sesamia inferens). Thus, introduction of an insecticidal proteinase inhibitor gene into cereal plants can be used as a general strategy for control of insect pests.

Animals↗
PubMed1995

DNA sequence of tail fiber genes of coliphage 186 and evidence for a common ancestor shared by dsDNA phage fiber genes.

We present here the nucleotide sequence of the tail fiber genes of phage 186. Marker rescue was used to associate an open reading frame (ORF) of 462 codons with the previously known tail gene K. A downstream ORF, encoding a 166-amino-acid product, was designated orf 45. Comparative studies suggested that K encodes the tail fiber protein and that orf 45 encodes an assembly protein. K protein contains a succession of short amino acid sequences (motifs) that are homologous with sequences from the tail fiber proteins of unrelated bacteriophages. The fact that these sequence motifs are variously present in the tail fiber proteins of unrelated bacteriophages has been advanced as evidence for horizontal transfer in the evolution of the associated tail fiber genes. However, the fact that the order of the various motifs in the proteins is invariant emphasizes the probability that independent divergence from a common ancestor also played a major role in the evolution of the tail fiber genes.

Amino Acid Sequence↗
PubMed1995

Tail sheath and tail tube genes of the temperate coliphage 186.

The nucleotide sequence of the phage 186 genome from 46.3-52.5% was determined and was found to contain two open reading frames highly similar to the tail sheath gene FI and tail tube gene FII of phage P2. The reading frames were identified as genes J and I by marker rescue experiments. A late promoter pJ was identified by galK reporter and primer extension studies. Northern analysis suggested that the pJ transcript predominantly terminated immediately after gene I, although some transcripts could extend to the terminator tB at 67.3%.

Amino Acid Sequence↗
PubMed1995

Trichodiene synthase. Substrate specificity and inhibition.

The substrate specificity of the sesquiterpene synthase trichodiene synthase was examined by determining the Vmax and Km parameters for the natural substrate, trans,trans-farnesyl diphosphate (1), its stereoisomer, cis,trans-farnesyl diphosphate, and the tertiary allylic isomer, (3R)-nerolidyl diphosphate (3), using both the native fungal and recombinant enzymes. A series of farnesyl diphosphate analogs, 15, 16, 20, 7, 8, and 9, was also tested as inhibitors of trichodiene synthase. 10-Fluorofarnesyl diphosphate (15) was the most effective competitive inhibitor, with a K1 of 16 nM compared to the Km for 1 of 87 nM, while the ether analog of farnesyl diphosphate, 8, an extremely potent inhibitor of squalene synthase, showed only modest inhibition of trichodiene synthase, with a K1/Km of 70.

Binding, Competitive↗
PubMed1995

Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Differences in the chemical reactivity of individual molecules of an enzyme.

Much attention has been focused recently on the detection and physical characterization of individual molecules. Using such methods to study the chemical properties, such as reactivity, of single molecules offers the potential to investigate how these might vary from molecule to molecule, and for individual molecules as a function of time. The complex structures of biomolecules such as enzymes make them particularly attractive targets for studying how subtle changes or differences at the molecular level might influence chemical reactivity. We have shown previously that very small (zeptomole) amounts of enzymes can be studied using a fluorescence microassay; single enzyme molecules have also been detected in oil-dispersed droplets by fluorescence microscopy. Here we report the observation of reactions of individual molecules of lactate dehydrogenase (LDH-1), which produces NADH from lactate and nicotinamide adenine dinucleotide (NAD+). When they are present at very low concentrations in a narrow capillary, each enzyme molecule produces a discrete zone of NADH; these can be manipulated electrophoretically and monitored by fluorescence spectroscopy. We find that the activity of individual electrophoretically pure enzyme molecules can vary by up to a factor of four, and that these activities remain unchanged over a two-hour period. We suggest that the origin of the activity differences may lie in the presence of several stable forms of the enzyme.

Enzyme Stability↗
PubMed1995

Analysis of high-frequency signal-averaged ECG measurements.

Analysis of high frequency (150-250 Hz) in the signal-averaged electrocardiogram (SAECG) is one of the emerging methods for detecting vessel patency in acute myocardial infarction following thrombolytic therapy and angioplasty. Root-mean-square voltage (RMSV) of the filtered QRS has been used in earlier studies to detect reperfusion; however, previous analysis indicated that RMSV is sensitive to residual noise in the SAECG and errors in QRS delineation (onset/offset). A new measurement is proposed, high-frequency energy (HFQE), and the robustness of the RMSV and HFQE was evaluated for simulated errors in QRS delineation. In this study, two measures (RMSV and HFQE) were tested on 24 control subjects and 21 patients undergoing thrombolytic therapy. Results indicate that unfiltered QRS duration is more stable than filtered QRS duration for the control subjects and patients and that HFQE had less fluctuation than RMSV in thrombolytic therapy patients. In the control group, HFQE was sensitive to the amplitude variation of the filtered SAECG. Therefore, another new measurement is proposed high-frequency integral of absolute value (HFAV), for reducing the sensitivity to amplitude changes in the filtered SAECG. This new feature was tested on control subjects and was found to be more stable than HFQE. In the thrombolitic therapy group, HFAV provided similar information as HFQE. These three measurements-RMSV, HFQE, and HFAV-provide a comprehensive analysis of the high-frequency SAECG for detecting vessel patency and reocclusion. Relative merits of these measures need to be evaluated on a larger database of patients undergoing thrombolysis and angioplasty for acute myocardial infarction.

Angioplasty, Balloon, Coronary↗