Biomedical subjects

M S Li

Publications and source records attributed to M S Li.

At least 19 recordsLinked to original sources

PubMed2002

Isolation and characterization of a strain of Bacillus thuringiensis ssp. kurstaki containing a new delta-endotoxin gene.

A strain of Bacillus thuringiensis that showed significantly high toxicity to Plutella xylostella and Spodoptera exigua was isolated from a Korean soil sample and characterized. The isolate, named B. thuringiensis K1, was determined to belong to ssp. kurstaki (H3a3b3c) type by an H antisera agglutination test and produced bipyramidal inclusions. Plasmid pattern of K1 was different from that of the reference strain, ssp. kurstaki HD-1, but the parasporal inclusion protein profile of K1 had two major bands that were similar in size to those of ssp. kurstaki HD-1. To verify the delta-endotoxin gene types of K1, PCR analysis with specific cry gene primers was performed to show that K1 contained a new cry gene in addition to cry1Aa, cry1Ab, cry1Ac, cry1E and cry2 genes. PCR-amplified region of the new cry gene, cryX, showed 79% similarity to cry1Fa1 gene (GenBank Accession No. M63897). In an insect toxicity assay, K1 had higher toxicity against Plutella xylostella and S. exigua than ssp. kurstaki HD-1.

Animals↗

PubMed2001

Influence of the yihE gene of Shigella flexneri on global gene expression: on analysis using DNA arrays.

Inactivation of dsbA (disulfide bond formation), either by an insertion (Sh4, dsbA::kan) or by alteration of the active site (Sh42, dsbA33G), renders Shigella flexneri avirulent. However, Sh4 and Sh42 behave differently in many ways in vitro and in vivo. A gene of unknown function, yihE, up-stream and cotranscribed with dsbA, is thought to differentiate Sh4 and Sh42 as the kan insertion may result in a truncated unstable yihE-dsbA mRNA in Sh4. To gain insight into the function of yihE, DNA array hybridization was performed to study the genomic expression in Sh4, Sh42, and a newly constructed yihE mutant (Sh54). Compared to the wild-type, M90TS, Sh4, and Sh54 demonstrated significantly changed transcription levels of about 100 genes, of which many involved in energy metabolism and stress response were down- and up-regulated, respectively. In contrast, Sh42 showed altered transcription levels of only 20 genes. The results argue that yihE is principally responsible for the changed genomic expression in Sh4 and Sh54. Given the fact that the transcription of yihE-dsbA is regulated by the CpxRA two-component signal transduction system, yihE is probably involved in the extracytoplasmic stress response in a manor deserving further studies.

Bacterial Proteins↗

PubMed2001

Aging effect in ceramic superconductors.

A three-dimensional lattice of the Josephson junctions with a finite self-conductance is employed to model ceramic superconductors. By using Monte Carlo simulations it is shown that the aging disappears in the strong screening limit. In the weak screening regime, aging is present even at low temperatures. For intermediate values of the self-inductance, aging occurs in an intermediate temperature interval but is suppressed entirely at high and low temperatures. Our results are in good agreement with experiments.

Journal Article↗

PubMed2001

Fluorescent microsphere-based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification.

Large-scale human genotyping requires technologies with a minimal number of steps, high accuracy, and the ability to automate at a reasonable cost. In this regard, we have developed a rapid, cost-effective readout method for single nucleotide polymorphism (SNP) genotyping that combines an easily automatable single-tube allele-specific primer extension (ASPE) with an efficient high throughput flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs an ASPE reaction using PCR-derived target DNA containing the SNP and a pair of synthetic complementary capture probes that differ at their 3' end-nucleotide defining the alleles. Each capture probe has been synthesized to contain a unique 25-nucleotide identifying sequence (ZipCode) at its 5' end. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to biotin-labeled ASPE reaction products, sequestering them for flow cytometric analysis. ASPE offers both an advantage of streamlining the SNP analysis protocol and an ability to perform multiplex SNP analysis on any mixture of allelic variants. All steps of the assay are simple additions of the solutions, incubations, and washes. This technique was used to assay 15 multiplexed SNPs on human chromosome 12 from 96 patients. Comparison of the microsphere-based ASPE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.2% agreement in genotype assignments. In addition, the microsphere-based multiplex SNPs assay system was adapted for the identification of bacterial samples by both ASPE and single base chain extension (SBCE) assays. A series of probes designed for different variable sites of bacterial 16S rDNA permitted multiplex analysis and generated species- or genus-specific patterns. Seventeen bacterial species representing a broad range of gram-negative and gram-positive bacteria were analyzed within 16 variable sites of 16S rDNA sequence. The results were consistent with the published sequences and confirmed by direct DNA sequencing.

Alleles↗

PubMed2001

Molecular cloning of a new crystal protein gene cry1Af1 of Bacillus thuringiensis NT0423 from Korean sericultural farms.

A new cry1Ab-type gene encoding the 130 kDa protein of Bacillus thuringiensis NT0423 bipyramidal crystals was cloned, sequenced, and expressed in a crystal-negative B. thuringiensis host. Hybridization experiments revealed that the crystal protein gene is located on a 44 MDa plasmid of B. thuringiensis NT0423. A strong positive signal detected on the 6.6 kb HindIII fragment from B. thuringiensis NT0423 plasmid DNA was cloned and sequenced. The cry1Ab-type gene, designated cry1Af1, consisted of open reading frame of 3453 bp, encoding a protein of 1151 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 130,215 Da. With both Bt I and Br II promoter sequences were found, the B. thuringiensis NT0423 crystal protein gene promoter closely aligned with those of cry1A-type crystal protein gene. When compared with known sequences of other Cry and Cyt proteins, the Cry1Af1 protein showed maximum 93% sequence identity to Cry1Ab protein of B. thuringiensis subsp. kurstaki. The expressed Cry1Af1 protein in a crystal-negative B. thuringiensis host appears to have strong insecticidal activity against lepidopteran larvae (Plutella xylostella). Crystals containing Cry1Af1 were about six times more toxic than the wild-type crystals of B. thuringiensis NT0423.

Agriculture↗

PubMed2001

Flow cytometric platform for high-throughput single nucleotide polymorphism analysis.

We have developed a rapid, cost-effective, high-throughput readout for single nucleotide polymorphism (SNP) genotyping using flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs a PCR-derived target DNA containing the SNP, a synthetic SNP-complementary ZipCode-bearing capture probe, a fluorescent reporter molecule, and a thermophilic DNA polymerase. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to the reaction products and sequestered them for flow cytometric analysis. The single base chain extension (SBCE) reaction was used to assay 20 multiplexed SNPs for 633 patients in 96-well format. Comparison of the microsphere-based SBCE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.3% agreement in genotype assignments. Substitution of direct-labeled R6G dideoxynucleotide with indirect-labeled phycoerythrin dideoxynucleotide enhanced signal five- to tenfold while maintaining low noise levels. A new assay based on allele-specific primer extension (ASPE) was validated on a set of 15 multiplexed SNPs for 96 patients. ASPE offers both the advantage of streamlining the SNP analysis protocol and the ability to perform multiplex SNP analysis on any mixture of allelic variants.

Flow Cytometry↗

PubMed2000

Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry.

BACKGROUND: We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres. METHODS: A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype. RESULTS: Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases. CONCLUSIONS: Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.

Chromosomes, Human, Pair 19↗

PubMed2000

Immunological analysis of the tegument phosphoprotein ppUL83 of human cytomegalovirus.

Immunological properties of the tegument phosphoprotein, ppUL83, of human cytomegalovirus (HCMV), expressed using a replication deficient recombinant adenovirus vector (RAd83) are described. The initial characterisation of this protein was carried out by immunofluorescence (IF), immunoprecipitation (RIP) and immunoblotting using nine mouse monoclonal antibodies (Mabs) directed against five linear and four conformational epitopes of ppUL83. The reactivity of the recombinant protein with the Mabs was similar to that observed with native ppUL83, although, the kinetics of its expression was in agreement with expression derived from the HCMV major immediate early promoter (MIEP). The recombinant antigen was used successfully in an Enzyme Immunoassay (EIA) for the detection of IgG class antibodies in 171 sequential sera taken from 21 heart transplant recipients. Comparison of HCMV-infected and RAd83-infected cell extracts in this experiment showed that recombinant antigen could substitute whole virus extracts as a single well-characterised protein in EIA. Serum IgG avidity measurements, using the recombinant ppUL83, differentiated between primary and past HCMV infections in the population studied.

Adenoviruses, Human↗

PubMed2000

A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension.

A rapid, high throughput readout for single-nucleotide polymorphism (SNP) analysis was developed employing single base chain extension and cytometric analysis of an array of fluorescent microspheres. An array of fluorescent microspheres was coupled with uniquely identifying sequences, termed complementary ZipCodes (cZipCodes), which allowed for multiplexing possibilities. For a given assay, querying a polymorphic base involved extending an oligonucleotide containing both a ZipCode and a SNP-specific sequence with a DNA polymerase and a pair of fluoresceinated dideoxynucleotides. To capture the reaction products for analysis, the ZipCode portion of the oligonucleotide was hybridized with its cZipCodes on the microsphere. Flow cytometry was used for microsphere decoding and SNP typing by detecting the fluorescein label captured on the microspheres. In addition to multiplexing capability, the ZipCode system allows multiple sets of SNPs to be analyzed by a limited set of cZipCode-attached microspheres. A standard set of non-cross reactive ZipCodes was established experimentally and the accuracy of the system was validated by comparison with genotypes determined by other technologies. From a total of 58 SNPs, 55 SNPs were successfully analyzed in the first pass using this assay format and all 181 genotypes across the 55 SNPs were correct. These data demonstrate that the microsphere-based single base chain extension (SBCE) method is a sensitive and reliable assay. It can be readily adapted to an automated, high-throughput genotyping system. [Primer sequences used in this study are available as online supplementary materials at www.genome.org.]

DNA, Complementary↗

PubMed2000

Dynamical chaos and power spectra in toy models of heteropolymers and proteins.

The dynamical chaos in Lennard-Jones toy models of heteropolymers is studied by molecular dynamics simulations. It is shown that two nearby trajectories quickly diverge from each other if the heteropolymer corresponds to a random sequence. For good folders, on the other hand, two nearby trajectories may initially move apart but eventually they come together. Thus good folders are intrinsically nonchaotic. A choice of a distance of the initial conformation from the native state affects the way in which a separation between the twin trajectories behaves in time. This observation allows one to determine the size of a folding funnel in good folders. We study the energy landscapes of the toy models by determining the power spectra and fractal characteristics of the dependence of the potential energy on time. For good folders, folding and unfolding trajectories have distinctly different correlated behaviors at low frequencies.

Computer Simulation↗

PubMed1999

Improved recovery of Mycobacterium tuberculosis from pleural aspirates: bedside inoculation, heparinized containers and liquid culture media.

The effects of bedside inoculation, heparinized containers and liquid culture media on the recovery of Mycobacterium tuberculosis from pleural aspirates were evaluated in this study. Of 155 patients, 63 were diagnosed to have pleural effusion tuberculous in origin. The overall recovery of M. tuberculosis was 57.1%. Bedside inoculation of the specimens produced a significantly higher yield than laboratory inoculation using non-heparinized specimens. When the pleural aspirates were transported in heparinized containers, the recovery rate was comparable to that from bedside inoculation, but lower when non-heparinized containers were used. No significant difference was found in recovery rate between the two liquid media, but the rate was significantly higher with the use of liquid media than conventional solid media. Thus, bedside inoculation of pleural aspirates, use of heparinized containers for transport for delayed inoculation in the laboratory and use of liquid culture media are recommended.

Anticoagulants↗

PubMed1999

Protective effect of allium vegetables against both esophageal and stomach cancer: a simultaneous case-referent study of a high-epidemic area in Jiangsu Province, China.

To study the relation between allium vegetable intake and cancer of the esophagus (EC) and stomach (SC) in Yangzhong city, which is one of the highest-risk areas for these cancers in Jiangsu province, China, a simultaneous case-referent study was conducted using histopathologically confirmed cases (EC: n = 81, SC: n = 153) and population-based referents (n = 234). A questionnaire was used to collect information on the general status of subjects, their dietary habits, frequency intake of allium vegetables and other foods, tea consumption, smoking and alcohol drinking. The odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by a multiple logistic regression model. The results showed that frequent intake of allium vegetables (including garlic, onion, Welsh onion and Chinese chives), raw vegetables, tomatoes and snap beans, and tea consumption were inversely associated with the risk for EC and SC. In the highest consumption category (> or = 1 time/week) of garlic, onion, Welsh onion and Chinese chives, the adjusted ORs compared with the lowest category (< 1 time/month) were 0.30 (CI = 0.19-0.47), 0.25 (CI = 0.11-0.54), 0.15 (CI = 0.08-0.26), and 0.57 (CI = 0.23-1.42) for EC, and 0.31 (CI = 0.22-0.44), 0.17 (CI = 0.08-0.36), 0.22 (CI = 0.15-0.31) and 0.40 (CI = 0.17-0.94) for SC, respectively. The main results in the present study suggested that allium vegetables, like raw vegetables, may have an important protecting effect against not only stomach cancer, but also esophageal cancer.

Adult↗

PubMed1999

Comparative study of lifestyles of residents in high and low risk areas for gastric cancer in Jiangsu Province, China; with special reference to allium vegetables.

There is a low risk area for gastric cancer in Jiangsu Province, China, where people frequently consume raw allium vegetables. As a first step epidemiological study to clarify the factors involved in the low incidence of gastric cancer, we conducted a comparative study of the ecological factors in a high risk area (HRA), Yangzhong, and a low risk area (LRA), Pizhou, using a questionnaire. Subjects were selected from the general population according to age and sex, and comprised 414 residents of the HRA and 425 residents of the LRA. Ecological factors were compared for the two areas by Cochran-Mantel-Haenszel method, age-adjusted. Smoking and drinking habits were found to be more common in the LRA. On the other hand, allium vegetables were consumed in the LRA much more frequently, with high consumption of raw vegetables fruit, tomatoes, kidney beans and soybean products. People who consumed garlic en masse 3 times or more per week were 82% of men and 75% of women in the LRA, and 1% of men and women in the HRA. The results of the survey suggest that frequent consumption of allium vegetables, in addition to other anticancer foods, may be a factor in low mortality for gastric cancer.

Adult↗

PubMed1997

[Clinical and experiment study of cooling therapy on burned wound].

OBJECTIVE: To observe the subjective benefit of pain relief by cooling therapy when wound temperature drops to below 28 degrees C (threshold for pain). METHOD: 22 patients with partial-thickness burns who received cooling therapy showed 2 days early healing as compared with untreated burn wounds. As a first aid measure, prolonged cooling therapy (up to 30') was recommended. In experimental study, the protective effects with cooling therapy were observed in wistar rats with deep second degree burns. RESULTS: Lower epithelial cell activity and higher schiff alkaline values were seen in the wound skin tissue in the non cooling therapy than in early cooling therapy groups and healthy controls. The groups with early cooling therapy (at once, 10', 20' after scalded injuries) the epithelial cell activity maintained at 83%, 80%, 65%. CONCLUSION: The results suggest that cooling therapy can protect the epithelial cell activity to some extent after scalded injuries which may be associated with inhibition of lipid peroxide.

Adult↗

PubMed1996

The Vpr protein of human immunodeficiency virus type 1 binds to nucleocapsid protein p7 in vitro.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) is incorporated into the virion by the Gag polyprotein precursor Pr55gag. The importance of the p6gag sequence at the C-terminal end of Pr55gag has a crucial role in Vpr incorporation. To identify the Gag sequences directly involved in Vpr binding, we compared the Vpr binding affinities of the 71 amino acid nucleocapsid protein p7, the C-terminal peptide (35-71) p7C and p6gag by affinity chromatography. p7 and p7C have the strongest Vpr binding activities compared to p6gag. These results suggest that the nucleocapsid protein and its C-terminal domain may be important for the incorporation of Vpr into the mature HIV-1 virion and the subsequent localisation of viral nucleic acid to the cell nucleus by Vpr.

Blotting, Western↗

PubMed1996

Topical glucocorticoids modulate the lesion interface after cerebral cortical stab wounds in adult rats.

A lesion interface, consisting of a glia limitans lined by a laminin-rich basal lamina and leptomeningeal cells, forms within 2-3 weeks after penetrating wounds to the adult mammalian central nervous system (CNS). This interface prevents the growth of axons across the lesion. We have examined the effects of topically applied steroids on the formation of such an interface after stab wounds to the adult rat cerebral cortex. Immediately after lesioning, the surface of cortex in the region of the wound was treated with a topical application of either 0.1% halcinonide or 0.05% betamethasone dipropionate or their respective placebos. Cryostat sections through the lesioned area were obtained 3 weeks later and assessed by immunofluorescence. Steroid treatment attenuated all components of the lesion. The continuous anti-laminin labeling along the lesion in untreated rats became patchy after steroid treatment. The number of leptomeningeal cells that infiltrated into the wound was reduced in the laminin-negative regions in steroid-treated rats. In addition, astrocytic processes in the laminin-negative regions after steroid treatment were loosely arranged, compared with the tightly packed parallel processes forming the glia limitans in laminin-positive regions in controls. The mechanism of steroid-mediated attenuation of the lesion interface was examined in vitro. Betamethasone but not halcinonide reduced laminin secretion slightly in leptomeningeal cell cultures, but both steroids reduced cell proliferation. These results suggest that steroids modulate the formation of the lesion interface after CNS injury, at least in part by decreasing leptomeningeal cell proliferation. Such modulation of the lesion interface by steroids or other agents may permit the growth of axons across the lesion site and thus could enhance the overall degree of axon regeneration if other factors such as neurotrophic support and neutralization of axon growth inhibitory molecules are optimized.

Animals↗

PubMed1995

Human eosinophil major basic protein, a mediator of allergic inflammation, is expressed by alternative splicing from two promoters.

Human eosinophil major basic protein (MBP) is one of the principal mediators of injury to parasites and tissues in allergic inflammation. MBP is stored in eosinophil crystalloid granules and released with other granule constituents during eosinophil action. Previous studies have identified an MBP gene promoter that generates a 1.0 kb mRNA transcript encoding MBP preproprotein which undergoes processing to the mature storage form. To investigate how the MBP gene is regulated, we have examined the identity and levels of the MBP transcripts both in precursor cells and in blood eosinophils. It was found that the gene was expressed from two upstream promoters, a distal promoter P1 in addition to the previously described promoter P2. Evidence for the second promoter was initially provided by isolation from a human HL-60 leukaemic cell cDNA library of a novel 1.6 kb MBP cDNA that was distinct from the known 1.0 kb cDNA. The complete nucleotide sequence of the 1.6 kb cDNA was determined, and showed that the two cDNAs had identical coding and 3' untranslated regions but differed in their 5' sequences. By isolating and sequencing MBP genomic clones from an arrayed chromosome 11 library, it was demonstrated that the MBP gene is composed of nine upstream exons and five coding exons. The 1.6 and 1.0 kb cDNAs arise by differential splicing of alternate MBP transcripts from promoters P1 and P2 respectively, located 32 kb apart in the genomic DNA. Primer extension analysis identified two transcription start sites at P1, neither associated with a typical TATA box motif. Northern blotting and reverse-transcription PCR analysis showed that the 1.0 kb mRNA was present at higher levels than the 1.6 kb species in immature cells including HL-60 and bone-marrow cells. By contrast, low levels of 1.6 kb mRNA transcripts predominated in differentiated blood eosinophils. The results are compatible with differential use of P1 and P2 promoters as a mechanism for regulation of MBP expression during eosinophil maturation.

Alternative Splicing↗

PubMed1995

Expression in Escherichia coli and purification of human eosinophil-derived neurotoxin with ribonuclease activity.

Eosinophil-derived neurotoxin (EDN) is a ribonuclease with neurotoxic and helminthotoxic properties. It is present in the crystalloid granules of human eosinophils. We report the expression and characterization of a functionally active recombinant human EDN using the pMAL-cRI expression system. A cDNA for mature EDN was obtained by PCR and inserted in pMAL-cRI downstream of the malE gene encoding maltose binding protein. Induction of the ptac promoter of the plasmid in Escherichia coli strain BL21(DE3) resulted in high level expression of soluble MAL-EDN fusion protein. Cleavage of affinity purified fusion protein with Factor Xa protease released recombinant EDN which comigrated with native EDN on SDS-polyacrylamide gels and cross-reacted with a polyclonal anti-EDN antiserum on Western blots. IN contrast to previous attempts at EDN expression, denatured and refolded EDN had ribonuclease activity and was prepared in microgram amounts. The availability of recombinant human EDN should facilitate studies of its structure and biological functions.

ATP-Binding Cassette Transporters↗