PubMed Health⌕ Search

Biomedical subjects

K Huang

Publications and source records attributed to K Huang.

At least 73 records · Page 4Linked to original sources
PubMed1999

Accurate quantitation of protein expression and site-specific phosphorylation.

A mass spectrometry-based method is described for simultaneous identification and quantitation of individual proteins and for determining changes in the levels of modifications at specific sites on individual proteins. Accurate quantitation is achieved through the use of whole-cell stable isotope labeling. This approach was applied to the detection of abundance differences of proteins present in wild-type versus mutant cell populations and to the identification of in vivo phosphorylation sites in the PAK-related yeast Ste20 protein kinase that depend specifically on the G1 cyclin Cln2. The present method is general and affords a quantitative description of cellular differences at the level of protein expression and modification, thus providing information that is critical to the understanding of complex biological phenomena.

Animals↗
PubMed1999

Study of erythrocyte sedimentation behavior by piezoelectric crystal impedance sensor.

Based on the impedance characteristic of erythrocytes at high frequency, the response of piezoelectric crystal impedance (PCI) sensor in the erythrocyte suspension was derived and verified experimentally. A method of using PCI sensor to investigate erythrocyte aggregation-sedimentation phenomenon was proposed. From the frequency response of the PCI sensor, the erythrocyte aggregation time and sedimentation rate could be obtained during erythrocyte aggregation and sedimentation. With the present method, the effects of the erythrocyte deformability, the osmotic pressure and the coexisting macromolecules on the erythrocyte sedimentation rate were studied. The results show that the PCI sensor possesses some advantages, such as good sensitivity, simplicity of use and no thermal effect for the impedance study of erythrocyte aggregation and sedimentation.

Blood Sedimentation↗
PubMed1999

Helix swapping between two alpha/beta barrels: crystal structure of phosphoenolpyruvate mutase with bound Mg(2+)-oxalate.

BACKGROUND: Phosphonate compounds are important secondary metabolites in nature and, when linked to macromolecules in eukaryotes, they might play a role in cell signaling. The first obligatory step in the biosynthesis of phosphonates is the formation of a carbon-phosphorus bond by converting phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr), a reaction that is catalyzed by PEP mutase. The PEP mutase functions as a tetramer and requires magnesium ions (Mg2+). RESULTS: The crystal structure of PEP mutase from the mollusk Mytilus edulis, bound to the inhibitor Mg(2+)-oxalate, has been determined using multiwavelength anomalous diffraction, exploiting the selenium absorption edge of a selenomethionine-containing protein. The structure has been refined at 1.8 A resolution. PEP mutase adopts a modified alpha/beta barrel fold, in which the eighth alpha helix projects away from the alpha/beta barrel instead of packing against the beta sheet. A tightly associated dimer is formed, such that the two eighth helices are swapped, each packing against the beta sheet of the neighboring molecule. A dimer of dimers further associates into a tetramer. Mg(2+)-oxalate is buried close to the center of the barrel, at the C-terminal ends of the beta strands. CONCLUSIONS: The tetramer observed in the crystal is likely to be physiologically relevant. Because the Mg(2+)-oxalate is inaccessible to solvent, substrate binding and dissociation might be accompanied by conformational changes. A mechanism involving a phosphoenzyme intermediate is proposed, with Asp58 acting as the nucleophilic entity that accepts and delivers the phosphoryl group. The active-site architecture and the chemistry performed by PEP mutase are different from other alpha/beta-barrel proteins that bind pyruvate or PEP, thus the enzyme might represent a new family of alpha/beta-barrel proteins.

Animals↗
PubMed1999

Pharmacokinetics and tissue disposition in monkeys of an antisense oligonucleotide inhibitor of Ha-ras encapsulated in stealth liposomes.

PURPOSE: This study examined the pharmacokinetics and tissue distribution of an antisense oligonucleotide ISIS 2503, formulated in stealth (pegylated) liposomes (encapsulated) or in phosphate-buffered saline (unencapsulated). METHODS: Encapsulated or unencapsulated ISIS 2503 was administered to rhesus monkeys by intravenous infusion. The concentrations of ISIS 2503 and metabolites in blood, plasma, and tissue samples were determined by capillary gel electrophoresis. RESULTS: Plasma concentrations of encapsulated ISIS 2503 decreased mono-exponentially after infusion with a mean half-life of 57.8 hours. In contrast, the concentration of unencapsulated ISIS 2503 in plasma decreased rapidly with a mean half-life of 1.07 hours. Both encapsulated and unencapsulated ISIS 2503 distributed widely into tissues. Encapsulated ISIS 2503 distributed primarily to the reticulo-endothelial system and there were few metabolites observed. In contrast, unencapsulated ISIS 2503 distributed rapidly to tissue with highest concentration seen in kidney and liver. Nuclease-mediated metabolism was extensive for unencapsulated oligonucleotide in plasma and tissues. CONCLUSIONS: The data suggest that stealth liposomes protect ISIS 2503 from nucleases in blood and tissues, slow tissue uptake, and slow the rate of clearance from the systemic circulation. These attributes may make these formulations attractive for delivering oligonucleotides to sites with increased vasculature permeability such as tumors or sites of inflammation.

Animals↗
PubMed1999

Phosphoinositide 3 kinase is critical for survival, mitogenesis and migration but not for differentiation of endothelial cells.

Angiogenesis involves endothelial cell invasion and migration into the surrounding tissue where cells differentiate, to form new lumen-containing vessels. We have investigated the role of phosphoinositide 3-kinase (PI3-kinase) in vascular endothelial growth factor (VEGF)- and fibroblast growth factor (FGF)-induced angiogenesis. Angiogenesis in vivo in chick embryos was inhibited by treatment with the PI3-kinase inhibitors wortmannin and LY294002. Stimulation of primary bovine capillary endothelial (BCE) cells with FGF-2, VEGF-A(165), or a combination of the two induced PI3-kinase activity in vitro and subsequent activation of the serine/threonine kinase Akt. The combination of FGF-2 and VEGF-A(165) led to an additive response. Activation of PI3-kinase was strictly required for FGF-2- and VEGF-A(165)-induced migration and DNA synthesis of BCE cells. Tubular morphogenesis was unaffected by treatment with wortmannin or LY294002, but survival of the tubular structures was dependent on PI3-kinase activity. VEGF-A(165) and FGF-2 induced increased stability of the tubular structures in a synergistic manner. These data indicate that PI3-kinase activity is required for migration, mitogenicity and survival but not for differentiation of endothelial cells during angiogenesis.

Journal Article↗
PubMed1999

The DNA helicase activity of BLM is necessary for the correction of the genomic instability of bloom syndrome cells.

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLM encodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of a Saccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern.

Adenosine Triphosphatases↗
PubMed1999

The influence of C-terminal extension on the structure of the "J-domain" in E. coli DnaJ.

Two different recombinant constructs of the N-terminal domain in Escherichia coli DnaJ were uniformly labeled with nitrogen-15 and carbon-13. One, DnaJ(1-78), contains the complete "J-domain," and the other, DnaJ(1-104), contains both the "J-domain" and a conserved "G/F" extension at the C-terminus. The three-dimensional structures of these proteins have been determined by heteronuclear NMR experiments. In both proteins the "J-domain" adopts a compact structure consisting of a helix-turn-helix-loop-helix-turn-helix motif. In contrast, the "G/F" region in DnaJ(1-104) does not fold into a well-defined structure. Nevertheless, the "G/F" region has been found to have an effect on the packing of the helices in the "J-domain" in DnaJ(1-104). Particularly, the interhelical angles between Helix IV and other helices are significantly different in the two structures. In addition, there are some local conformational changes in the loop region connecting the two central helices. These structural differences in the "J-domain" in the presence of the "G/F" region may be related to the observation that DnaJ (1-78) is incapable of stimulating the ATPase activity of the molecular chaperone protein DnaK despite evidence that sites mediating the binding of DnaJ to DnaK are located in the 1-78 segment.

Amino Acid Sequence↗
PubMed1999

Characterization of methylglyoxal synthase from Clostridium acetobutylicum ATCC 824 and its use in the formation of 1, 2-propanediol.

A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The Km and Vmax values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min(-1) microgram(-1), respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.

Amino Acid Sequence↗
PubMed1999

Apoptosis induced by infection of primary brain cultures with diverse human immunodeficiency virus type 1 isolates: evidence for a role of the envelope.

Apoptosis of neurons and astrocytes is induced by human immunodeficiency type 1 (HIV-1) infection in vitro and has been demonstrated in brain tissue from patients with AIDS. We analyzed a panel of diverse HIV-1 primary isolates for the ability to replicate and induce neuronal and astrocyte apoptosis in primary human brain cultures. Apoptosis was induced three- to eightfold by infection with the blood-derived HIV-1 isolates 89.6, SG3, and ADA. In contrast, the brain-derived HIV-1 isolates YU2, JRFL, DS-br, RC-br, and KJ-br did not induce significant levels of apoptosis. The ability of HIV-1 isolates to induce apoptosis was independent of their replication capacity. Studies of recombinant chimeras between the SG3 and YU2 viruses showed that replacement of the YU2 Env with the SG3 Env was sufficient to confer the ability to induce apoptosis to the YU2 virus. Replacement of the Env V3 regions alone largely conferred the phenotypes of the parental clones. The SG3 Env used CXCR4 and CCR3 as coreceptors for virus entry, whereas YU2 used CCR5 and CCR3. The V3 regions of SG3 and YU2 conferred the ability to use CXCR4 and CCR5, respectively. In contrast, the 3' region of Env, particularly the C3V4 region, was required in conjunction with the V3 region for efficient use of CCR3. These results provide evidence that Env is a major determinant of neurodegenerative mechanisms associated with HIV-1 infection in vitro and raise the possibility that blood-derived viruses which emerge during the late stages of disease may affect disease progression in the central nervous system.

Animals↗
PubMed1999

Cloning and characterization of promoter of the mouse mafB gene.

The MafB transcription factor plays a pivotal role in controlling the development and differentiation. The author reports the isolation and analysis of genomic clone of the mouse mafB gene. The gene lacks intron structure, at least, within its coding and 5'-untranslated sequences that are similar to the chicken mafB gene. RNA protection analysis determined one transcription initiation site of the gene at 389-bp upstream from the translation initiation site. Sequence analysis showed that the 5'-flanking region upstream to the ATG codon did not contain a conventional TATA box. A TATA-like sequence (5'-GATAAAA-3') and an inverted CCAAT-box (5'-ATTGG-3') were found to be located at nucleotide -31 and -86, referring to the transcription initiation site, respectively. Upstream to these sequences, there were several potential regulatory elements, including two GC-boxes (5'-GGGCGG-3': from -148 to -143; and from -123 to -118), and a palindromic sequence (5'-GTCAGCTGAC-3': from -164 to -155) which contained two halves-MARE (Maf recognition element), 5'-GCTGAC-3', and an E-box (5'-CAGCTG-3'). Promoter activity of the 5'-flanking region was analyzed by reporter transfection assay, which suggested that these segments were an important transcriptional activator. It was also suggested that MyoD transactivated the mouse mafB promoter and this gene was positively autoregulated by its product, MafB.

Animals↗
PubMed1999

Blocking L-calcium current by l-tetrahydropalmatine in single ventricular myocyte of guinea pigs.

AIM: To study the effect of l-tetrahydropalmatine (l-THP) on L-type calcium channel. METHODS: Patch clamp technique (whole cell recording) was used to record L-Ca2+ current in single cardiac myocyte. RESULTS: 1) l-THP 1, 10, and 100 micromol.L-1 reduced ICa-max from (999 +/- 93) pA to (700 +/- 111) pA, (582 +/- 66) pA, and (420 +/- 112) pA (n = 6, P < 0.01), respectively. 2) l-THP reduced the voltage at half-maximal inactivation (V1/2) of L-Ca2+ channel to more negative potentials by 9 mV (n = 5, P < 0.05). 3) l-THP caused both tonic and use-dependent reduction of Ca2+ current. Tonic block of l-THP on Ca2+ current was 46% +/- 8% (n = 6, P < 0.01). The degree of use dependent blocking was 13.5% +/- 2.4% (n = 6, P < 0.05) at 1 Hz, the degree increased to 44% +/- 5% (n = 6, P < 0.01) at 3 Hz. 4) l-THP delayed half-recovery time of Ca2+ channel recovery from inactivity from (94 +/- 39) ms to (170 +/- 42) ms(n = 6, P < 0.01). CONCLUSION: l-THP has a moderate inhibitory effect on L-Ca2+ current.

Animals↗
PubMed1999

Amplified fragment length polymorphism analysis on D5S436 locus and its application to linkage analysis in gene diagnosis of asthma.

OBJECTIVES: To detect the polymorphism of D5S436 locus, and to carry out an amplified fragment length polymorphism (Amp-FLP) genotyping linkage analysis in asthma families. METHODS: Allele and genotype frequencies of the high polymorphic D5S436 locus were determined in 92 unrelated Chinese individuals by using polymerase chain reaction (PCR). The amplified fragments were separated by denaturing polyacrylamide gel electrophoresis and silver staining techniques. RESULTS: Eight alleles and 22 genotypes were observed in this population. The heterozygosity was 78.26%. The polymorphism information content (PIC) was 0.76. Amp-FLP genotyping linkage analyses were carried out in four asthma families, demonstrating that the locus was inherited according to Mendel's law and had a clear result. CONCLUSION: The PIC of D5S436 is high in Chinese individuals, so it could be used as a genetic marker of asthma in Chinese and might be useful in the gene diagnosis of asthma.

Alleles↗
PubMed1999

[Linked marker analysis of bronchial asthma in China].

OBJECTIVE: To study the clinical application value of genetic linkage analysis for bronchial asthma with two STRs-D5S436 and D5S658. METHODS: Polymorphism of the two STRs were amplified by polymerase chain reaction, and the alleles identification were performed with denaturing polyacrylamide gel electrophoresis and silver-staining techniques. RESULTS: In 92 unrelated Chinese individuals, 8 alleles and 22 phenotypes in D5S436, 9 alleles and 28 phenotypes in D5S658 were observed. The heterozygosities were 78% and 73% respectively. The polymorphism information contents (PIC) were 0.76 and 0.80 respectively. Linkage analysis showed linkage of asthma related phenotypes (including asthma) with D5S436 on chromosome 5q (lod = 2.490, theta = 0.1). CONCLUSIONS: D5S436 and D5S658 were good genetic markers. Linkage analysis demonstrates that locus on chromosome 5q31-33 maybe was linked to the development of asthma in Chinese population.

Adolescent↗
PubMed1999

[A study of airway reactivity and nature of chronic asthmatic bronchitis].

OBJECTIVE: To observe the airway reactivity with methacholine provocation and exercise challenge of chronic asthmatic bronchitis and to assess preliminarily the nature of chronic asthmatic bronchitis. METHODS: 42 patients with bronchial asthma, 29 with simple chronic bronchitis and 27 with chronic asthmatic bronchitis were selected. The study protocol consisted of inhalation challenge with methacholine and exercise challenge. Count of blood eosinophil cells and level of blood total IgE were studied. Serum eosinophil cationic protein (ECP) concentration was measured in the asthmatic bronchitis group. RESULTS: The positive ratio to methacholine response was 77.8% in chronic asthmatic bronchitis group, whereas the ratios were 100.0% and 41.4% in bronchial asthma and simple chronic bronchitis groups respectively. According to the response to exercise challenge, patients with chronic asthmatic bronchitis were divided into positive and negative groups. The cumulative dose of methacholine producing a 20% fall in FEV(1) (PD(20)-FEV(1)) was different between the two groups. In the positive group (9 patients), none was negative with methacholine response and PD(20)-FEV(1) being similar to the bronchial asthma group. The blood total IgE and ECP levels were significantly higher in the positive than in the negative group. CONCLUSION: The characteristics of airway reactivity in chronic asthmatic bronchitis are midway between bronchial asthma and simple chronic bronchitis. The nature of chronic asthmatic bronchitis may be different in different patients.

Adult↗
PubMed1999

[Comparison of sensitivity of artesunate-sensitive and artesunate-resistant Plasmodium falciparum to chloroquine and amodiaquine].

AIM: To explore the in vitro sensitivity of artesunate-sensitive and -resistant Plasmodium falciparum to chloroquine and amodiaquine and to observe the effect of artesunate combined with chloroquine and artesunate combined with amodiaquine on artesunate-resistant P. falciparum. METHODS: The sensitivity of the artesunate-sensitive and -resistant P. falciparum to chloroquine and amodiaquine and their combination with artesunate was compared by using Rieckmann's in vitro micro-technique. RESULTS: The ID50 and ID95 values of chloroquine, amodiaquine and artesunete were 90.9, 50.9, 9.6 nmol/L and 320.0, 320.0, 40.0 nmol/L to the artesunate-sensitive P. falciparum respectively and were 112.0, 133.5, 85.1 nmol/L and 320.0, 320.0, 400.0 nmol/L to artesunate-resistant P. falciparum, respectively. When artesunate was combined with chloroquine, the ID50 values of the 2 drugs were 3.2 and 20.0 nmol/L to the artesunate-resistant P. falciparum. When artesunate was combined with amodiaquine, the ID95 values of the 2 drugs were 3.2 and 5.0 nmol/L to the artesunate-resistant P. falciparum, respectively. CONCLUSION: The artesunate-resistant P. falciparum has no cross resistance to chloroquine and amodiaquine, however, artesunate combined with chloroquine or amodiaquine exhibit an apparent synergistic effect in vitro.

Amodiaquine↗
PubMed1999

[Assay of sensitivity of Plasmodium falciparum to chloroquine, amodiaquine, piperaquine, mefloquine and quinine in Yunnan province].

AIM: To determine the sensitivity of P. falciparum to chloroquine, amodiaquine, piperaquine, mefloquine and quinine in Yunnan Province of China in 1992-1995. METHODS: Rieckmann's in vitro microtechnigue was used. The sensitivity of P. falciparum was tested to the above-mentioned antimalarials. RESULTS: The resistance rates of isolates of P. falciparum from the south, southeast and the west part of Yunnan to chloroquine and amodiaquine were 96.7% (29/30), 78.9% (30/38), 95.7% (22/23) and 100% (30/30), 85.3% (29/34), 8/9, respectively, with their corresponding ID50 of 125 nmol/L, 136 nmol/L and 176 nmol/L, and 52 nmol/L, 64 nmol/L and 72 nmol/L, respectively. All the isolates were sensitive to quinine and their ID50 were 480 nmol/L, 352 nmol/L, 608 nmol/L, respectively. The resistance rates of P. falciparum from the south part and southeast part of Yunnan to piperaquine were 96.4% (27/29), 72.9 (27/37), respectively, their ID50 were 320 nmol/L and 228 nmol/L; all the cases were sensitive to mefloqine, their ID50 were 68 nmol/L and 88 nmol/L. CONCLUSION: P. falciparum generally produces resistance to chloroquine, amodiaquine and piperaquine in Yunnan Province; the degree of resistance to chloroquine of P. falciparum from the west part of Yunnan were higher than the P. falciparum from the southeast part of Yunnan; all the isolates were sensitive to mefloquine and quinine in this region.

Amodiaquine↗