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Biomedical subjects

J Yan

Publications and source records attributed to J Yan.

At least 55 records · Page 3Linked to original sources
PubMed2002

Over-expression of beta-1,4-galactosyltransferase V increases the growth of astrocytoma cell line.

Our previous study showed that the gene expression of beta-1,4-galactosyltransferase V (beta-1,4-GalT V), preferentially galactosylating GlcNAc1-->6Man of oligosaccharides, increased in the process of astrocytoma progress, with the highest level in grade IV astrocytoma. To investigate the function of this beta-1,4-GalT in cell proliferation, the sense and antisense cDNA of beta-1,4-GalT V was constructed as pcDNA3-HA-GalT V and pcDNA3-anti-GalT V respectively and transfected into SHG cell, a kind of human astrocytoma cell line. The transfection was confirmed with Northern and Western blot assay. It was found that the growth of SHG/HA-GalT V in serum-containing medium was faster than that of mock-transfectant with the vector pcDNA3, whereas the growth of SHG/GalTV-AS was slower than that of mock-transfectant. GalTV-HA/SHG showed a stronger capability for colony formation than that of GalTV-AS/SHG as evaluated by anchorage-independent growth in soft agar assay. This result was consistent with that of the growth curve. By RCA-1 lectin assay, the galactosylation on the surface of GalTV-HA/SHG and SHG/GalTV-AS was stained stronger (P<0.001) and weaker (P<0.05) respectively compared with the mock transfectant. This indicates that beta-1,4-GalT V was involved in the malignant phenotype of astrocytoma cells, possibly causing the high galactosylation on the cell surface.

Astrocytoma↗
PubMed2002

Selection of phage-display peptides that bind specifically to the outer coat protein of Rice black streaked dwarf virus.

Several peptides that could bind specifically to the outer coat protein encoded by the S10 gene of Rice black streaked virus (RBSDV) were isolated from a phage-display random 12-mer peptide library. The sequence analysis showed that the amino acid motif (K)K**(*)P, the asterisk denoting any amino acid, might be the core sequence by which the peptides bind to the target protein. The peptide 1 that had a high affinity to RBSDV outer coat protein was synthesized by a chemical method and its fusion protein with glutathione-S-transferase (GST) was produced in an Escherichia coli expression system. The dot and Western blot analyses indicated that RBSDV could be detected with a high sensitivity in crude extracts of diseased plant leaves using a purified GST fusion protein. The circular dichroism (CD) spectroscopy revealed that the synthesized binding peptide but not a nonbinding peptide could bring about a marked change in the conformation of outer coat RBSDV protein. Since the protein functions only when it has correct conformation, the peptides binding specifically to it could possibly disturb the function of the virus outer coat protein and might be used to block the transmission pathway of the virus. Summing up, as these peptides showed a high specificity and sensitivity and diagnostic potential for RBSDV, they may represent the basis of a novel strategy for development of resistance to RBSDV.

Artificial Gene Fusion↗
PubMed2001

Further characterization and population data for the pentanucleotide STR polymorphism D10S2325.

Pentanucleotide tandem repeat markers are interesting for forensic sciences, because they may present less stutter on the electrophoretic pattern. We focused on the analysis of the DNA sequence for each allele at the pentanucleotide STR locus D10S2325 in order to understand their structures in the human genome and to construct human allelic ladder, which is necessary for forensic DNA typing. In order to evaluate the forensic applicability of D10S2325 and to construct a preliminary database, the genotype distributions and allele frequencies in three major ethnic groups were investigated. The population samples included Caucasians (Germans), Africans (African Americans), and Asians (Chinese). A total of 520 samples from unrelated individuals was analyzed by Amp-FLP. An example of each allele and new alleles were sequenced. Allele determination was carried out by comparison with a sequenced human allelic ladder made in-house. This pentanucleotide STR provided easily interpretable results. A total of 15 alleles was found in our population samples. Three new alleles were observed and named as alleles 19 and 21 based on the number of repeat motifs, while allele 19 can be divided further into two alleles, 19a and 19 according to analysis of the sequence. No evidence of deviation from Hardy-Weinberg equilibrium was observed. In 64 confirmed father/mother/child triplets no mutation event was observed. Using a maximum likelihood method, the mutation rate was indirectly estimated as 2.5 x 10(-5). These results suggest that D10S2325 is a useful marker for forensic casework and paternity analysis.

Black or African American↗
PubMed2001

The presence of 9-cis-beta-carotene in cytochrome b(6)f complex from spinach.

Cytochrome b(6)f complex with stoichiometrically bound beta-carotene molecule was purified from spinach chloroplasts. The configuration of this beta-carotene was studied by reversed-phase HPLC and resonance Raman spectroscopy. Both the absorption spectrum of this beta-carotene in dissociated state and the Raman spectrum in native state can be unambiguously assigned to a 9-cis configuration. This finding is in contrast to the predominantly all-trans isomers commonly found in membranes and protein-pigment complexes of chloroplasts, suggesting that the 9-cis-beta-carotene is an authentic component and may have a unique structural and functional role in cytochrome b(6)f complex.

Chromatography, High Pressure Liquid↗
PubMed2001

Corticofugal reorganization of the midbrain tonotopic map in mice.

Previous studies have indicated that frequency maps (tonotopies) in mammalian auditory brain centers are plastic. Here, we examined this plasticity in the mouse auditory midbrain through focal stimulation of the primary auditory cortex. Cortical activation shifted midbrain frequency tunings toward the best frequencies of the stimulated cortical neurons if these were either higher or lower than the cortical ones. Such corticofugal adjustments appear to minimize the difference between cortical and collicular frequency tuning within the critical bandwidths of the auditory system. Consequently, the neural representation is enhanced for the frequencies to which the cortical neurons were tuned. Our data suggest that the auditory cortex reorganizes midbrain tonotopy on the basis of which cortical frequencies are stimulated, mostly probably through corticofugal projections.

Acoustic Stimulation↗
PubMed2001

Theoretical study of opacity for a mixture of gold and gadolinium at a high temperature.

Using the detailed configuration accounting with the term structures treated by the unresolved transition array model, we have presented a method to calculate the spectral-resolved opacity for high temperature and density plasmas. Due to the fully relativistic treatment, incorporated with the quantum defect theory to handle the huge number of transition arrays from configurations with high principal quantum number, we can calculate the opacity of any medium- and high-Z plasmas conveniently. In the present work, the frequency-dependent opacity and the Rosseland mean opacity are calculated for a mixture of gold and gadolinium at a high temperature, 250 eV, and three densities, 0.1 g/cm(3), 1.0 g/cm(3), and 10.0 g/cm(3). Agreement between our theoretical results and experimental measurements and other theoretical simulations is obtained.

Journal Article↗
PubMed2001

Diagnosis of Duchenne dystrophy by enhanced detection of small mutations.

OBJECTIVE: To determine whether detection of small mutations of the dystrophin gene can be increased using an enhanced method of single-strand conformation polymorphism analysis. BACKGROUND: Usual methods of DNA analysis for Duchenne dystrophy cannot identify mutations in one-third of cases. Muscle biopsy, with its inherent risks and added liability for patients with Duchenne dystrophy, becomes the sole method of diagnosis. Even with a tissue diagnosis of dystrophin deficiency, many families are excluded from carrier detection and prenatal diagnosis. METHODS: Genomic DNA from a cohort of 93 patients with Duchenne dystrophy without identifiable gene mutations was screened for mutations. In each case, 22 kilobases of genomic DNA were scanned, including all 79 exons of the dystrophin gene, adjacent intronic regions, and six alternative exons 1. RESULTS: Sixty-eight (73%) had small mutations, including 34 nonsense mutations, 27 microdeletions and insertions, and 7 splice site mutations. No missense mutations were found. One nonsense mutation in exon 59 was detected in four patients. Most mutations were new; 54 of 62 different small mutations have not been reported. Mutations were found throughout the gene: 24% in the first quartile, 31% in the second, 16% in the third, and 29% in the fourth. CONCLUSIONS: A highly sensitive single-strand conformation polymorphism method substantially increased detection of small dystrophin gene mutations and made it possible to diagnose approximately 90% of patients with Duchenne dystrophy by DNA analysis. These findings, combined with cost savings and safety issues, provide compelling reasons to consider DNA analysis as the initial diagnostic test for the suspected dystrophin-deficient patient.

Adolescent↗
PubMed2001

[Significance of her2 oncogene expression in primary nasopharyngeal carcinoma].

OBJECTIVE: To investigate the status of her2 oncogene at DNA level and the molecular mechanism of overexpression of her2 oncoprotein in part of patients with nasopharyngeal carcinoma (NPC). METHODS: Forty-five cases of PNC tissue and fifteen cases of normal epithelial tissue adjacent to tumor tissue were examined for her 2 signal and centromere 17 signal. Interphase fluorescence in situ hybridization (FISH) was used to examine the copy number of her 2 and immunohistochemical (IHC) staining were used to analyze the copy number and protein expression of her2. RESULTS: There were 1.92% +/- 0.10% her2 signals per nucleus in normal epithelial tissue adjacent to tumor. 75.52 +/- 9.33% of the nuclei contained 2 her2 signals and 2 centromere 17 signals. No alteration in gene copy number was found in cancerous nuclei. However, IHC staining of the 45 tumors with antibody against her2 showed that overexpression of her2 oncoprotein occurred in 33% (15/45) of NPC. However, there was no significant correlation between her2 expression and sex, age, clinical staging, and clinical outcome of patients. CONCLUSION: The overexpression of her2 in NPC does not relate to her2 gene amplification. Immunohistochemistry may not be a good measure to examine her2 expression. The mechanism of her2 oncoprotein overexpression and its biological significance in the development of NPC remain to be elucidated.

Adult↗
PubMed2001

Retinoic acid promotes differentiation of trophoblast stem cells to a giant cell fate.

Trophoblast stem cell (TS cell) lines have the ability to differentiate into trophoblast subtypes in vitro and contribute to the formation of placenta in chimeras. In order to investigate the possible role of retinoic acid (RA) in placentation, we analyzed the effects of exogenous RA on TS cells in vitro and the developing ectoplacental cone in vivo. TS cells expressed all subtypes of the retinoid receptor family, with the exception of RARbeta, whose expression was stimulated in response to RA. TS cells treated with RA were compromised in their ability to proliferate and exhibited properties of differentiation into trophoblast giant cells. During TS cell differentiation into trophoblast subtypes induced by withdrawal of FGF4, RA treatment further illustrated its role in the specification of cell fate by the promotion of differentiation into giant cells and the suppression of spongiotrophoblast formation. Moreover, administration of RA during pregnancy resulted in the overabundance of giant cells at the expense of spongiotrophoblast cells. RA hereby acts as an extracellular signal whose potential function can be linked to specification events mediating trophoblast cell fate. Taken together with the spatial patterns of giant-cell formation and RA synthesis in vivo, these findings implicate a function for RA in giant-cell formation during placentation.

Animals↗
PubMed2001

An optimized PCR-based procedure for production of 13C/15N-labeled DNA.

We have substantially improved a procedure that we previously described for producing 13C/15N-labeled DNA (Chen et al., FEBS Lett. 436, 372-376, 1998) to provide an economical and straightforward approach to the preparation of labeled DNA. The conditions for the PCR reactions have been optimized to permit the use of low concentrations of the costly labeled dNTPs (50 microM for each). In addition, a rapid and high-yield purification procedure has been developed that allows us to obtain a high yield of very pure labeled DNA. These modifications to our original procedure permit us to obtain 1.9 mg of an 18 bp DNA oligomer from 20 mg of dNTPs (ca. 10% yield from the starting dNTPs). This is sufficient material for the preparation of 0.4 mM sample in a volume of 400 microl. In summary, this procedure is a cost-effective, time-efficient procedure for the production of labeled DNA for NMR studies.

Carbon Isotopes↗
PubMed2001

Ultrasensitive assay for three polyphenols (catechin, quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection.

The concentrations of three polyphenols ((+)-catechin, quercetin and trans-resveratrol) in blood serum, plasma and urine, as well as whole blood, have been measured after their oral and intragastric administration, respectively, to humans and rats. The method developed for this purpose utilized ethyl acetate extraction of 100 microl samples and their derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by gas-chromatographic analysis on a DB-5 column followed by mass selective detection employing two target ions and one qualifier ion for each compound. Total run time was 17 min with excellent resolution and linearity. The limits of detection (LOD) and quantitation (LOQ) were an order of magnitude less than for any previously published method, being 0.01 microg/l and 0.1 microg/l, respectively, for all compounds. Recovery at 1 microg/l and 10 microg/l was >80% in all instances but one, and was >90% in 50%. Imprecision was acceptable at 0.25 and 1.0 microg/l, concentrations below the LOQ of previous methods. Aglycones released from conjugates after hydrolysis were easily measurable. Optimal conditions for hydrolysis were established. After oral administration of the three polyphenols to humans, their conjugates vastly exceeded the concentrations of the aglycones in both plasma and urine. Concentrations peaked within 0.5-1.0 h in plasma and within 8 h in urine. During the first 24 h, 5.1% of the (+)-catechin and 24.6% of the trans-resveratrol given were recovered in the urine (free plus conjugated). This method can be proposed as the method of choice to assay these polyphenols and their conjugates in biological fluids.

Calibration↗
PubMed2001

Scanning of estrogen receptor alpha (ERalpha) and thyroid hormone receptor alpha (TRalpha) genes in patients with psychiatric diseases: four missense mutations identified in ERalpha gene.

Estrogen and thyroid hormones exert effects on growth, development, and differentiation of the nervous system. Hormone administration can lead to changes in behavior, suggesting that genetic variants of the estrogen receptor alpha (ERalpha) and the thyroid hormone receptor alpha (TRalpha) genes may predispose to psychiatric diseases. To investigate this possibility, regions of likely functional significance (all coding exons and flanking splice junctions) of the ERalpha and TRalpha genes were scanned in patients with schizophrenia (113), along with pilot studies in patients with bipolar illness (BPI), puerperal psychosis, autism, attention-deficit hyperactivity disorder (ADHD), and alcoholism. A total of 1.18 megabases of the ERalpha gene and 1.16 megabases of the TRalpha gene were scanned with Detection of Virtually All Mutations-SSCP (DOVAM-S), a method that detects virtually all mutations. Four missense mutations, seven silent mutations and one deletion were identified in the ERalpha gene, while only four silent mutations were present in the TRalpha gene. Two of the missense mutations in ERalpha are conserved in the six available mammalian and bird species (H6Y, K299R) and a third sequence variant (P146Q) is conserved in mammals, birds, and Xenopus laevis, hinting that these sequence changes will be of functional significance. These changes were found in one patient each with BPI, puerperal psychosis, and alcoholism, respectively. Analysis of the ERalpha and TRalpha genes in 240 subjects reveals that missense changes and splice site variants are uncommon (1.7% and 0%, respectively). Further analyses are necessary to determine if the missense mutations identified in this study are associated with predisposition or outcome for either psychiatric or nonpsychiatric diseases.

Alleles↗
PubMed2001

Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343.

Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phosphatidylinositol (PI) 3-kinase-dependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. We now show by in-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB- serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt.

Amino Acid Sequence↗
PubMed2001

Kinetic proofreading can explain the supression of supercoiling of circular DNA molecules by type-II topoisomerases.

The enzymes that pass DNA through DNA so as to remove entanglements, adenosine-triphosphate-hydrolyzing type-II topoisomerases, are able to suppress the probability of self-entanglements (knots) and mutual entanglements (links) between approximately 10 kb plasmids, well below the levels expected, given the assumption that the topoisomerases pass DNA segments at random by thermal motion. This implies that a 10-nm type-II topoisomerase can somehow sense the topology of a large DNA. We previously introduced a "kinetic proofreading" model which supposes the enzyme to require two successive collisions in order to allow exchange of DNA segments, and we showed how it could quantitatively explain the reduction in knotting and linking complexity. Here we show how the same model quantitatively explains the reduced variance of the double-helix linking number (supercoiling) distribution observed experimentally.

Binding Sites↗
PubMed2001

[Clinical usefulness of pulsatile bidirectional Glenn shunt].

OBJECTIVE: To evaluate the clinical usefulness of pulsatile bidirectional Glenn shunt. METHODS: Between June 1994 and January 2000, pulsatile bidirectional Glenn shunt was operated on 41 patients, 31 males and 10 females, aged 5 +/- 4 Y (7M approximately 16Y) and with the body surface area of 0.64 +/- 0.25 m(2) (0.35 approximately 1.48 m(2)), 35 operations being performed under hypothermia with cardiopulmonary bypass, and the other 6 operations being performed at ordinary temperature without cardiopulmonary bypass. The SaO(2) before operation was 70% +/- 11% (25% approximately 89%). The pulmonary pressure measured during operation was 12.5 mm Hg +/- 2.7 mm Hg (8 approximately 17 mm Hg). RESULTS: One patient died in the early postoperative period, three got chylothorax, and the others made uneventful recovery. This operation provides excellent early palliation of cyanosis. The average SaO(2) was 90% +/- 4% (77% approximately 96%) on discharge. The time of postoperative respirator usage duration was shorter in those patients who underwent operation at ordinary temperature without cardiopulmonary bypass. CONCLUSION: The pulsatile bidirectional Glenn shunt, easy to perform and with good early postoperative effects, is a useful procedure in the early management of patients with a functional univentricular heart.

Adolescent↗