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Biomedical subjects

Y Chen

Publications and source records attributed to Y Chen.

At least 793 records · Page 44Linked to original sources
PubMed2000

Lutein and zeaxanthin in the eyes, serum and diet of human subjects.

Inverse associations have been reported between the incidence of advanced, neovascular, age-related macular degeneration (AMD) and the combined lutein (L) and zeaxanthin (Z) intake in the diet, and L and Z concentration in the blood serum. We suggest that persons with high levels of L and Z in either the diet or serum would probably have, in addition, relatively high densities of these carotenoids in the macula, the so-called 'macular pigment'. Several lines of evidence point to a potential protective effect by the macular pigment against AMD. In this study we examined the relationship between dietary intake of L and Z using a food frequency questionnaire; concentration of L and Z in the serum, determined by high-performance liquid chromatography, and macular pigment optical density, obtained by flicker photometry. Nineteen subjects participated. We also analysed the serum and retinas, as autopsy samples, from 23 tissue donors in order to obtain the concentration of L and Z in these tissues. The results reveal positive, though weak, associations between dietary intake of L and Z and serum concentration of L and Z, and between serum concentration of L and Z and macular pigment density. We estimate that approximately half of the variability in the subjects' serum concentration of L and Z can be explained by their dietary intake of L and Z, and about one third of the variability in their macular pigment density can be attributed to their serum concentration of L and Z. These results, together with the reported associations between risk of AMD and dietary and serum L and Z, support the hypothesis that low concentrations of macular pigment may be associated with an increased risk of AMD.

Adolescent↗
PubMed2000

Fluorescence Excitation Spectrum of a (2)Pi(3/2)-(2)Pi(3/2) Transition of NiF.

In this study, a supersonic beam of NiF was produced by the reaction of SF(6) with a dc discharge-sputtering source of nickel atoms. The laser-induced fluorescence excitation spectrum of a (2)Pi(3/2)-(2)Pi(3/2) transition has been recorded in the range of 500-520 nm and rotational structure of 506.5-nm band analyzed under the 30 K rotational temperature. Our data are consistent with a (2)Pi(3/2) ground state for NiF. The lifetime of this band is measured. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Polymerization of deoxy-sickle cell hemoglobin in high-phosphate buffer.

Deoxy-sicklecell hemoglobin (HbS) polymerizes in 0.05 M phosphate buffer to form long helical fibers. The reaction typically occurs when the concentration of HbS is about 165 mg/ml. Polymerization produces a variety of polymorphic forms. The structure of the fibers can be probed by using site-directed mutants to examine the effect of altering the residues involved in intermolecular interactions. Polymerization can also be induced in the presence of 1.5 M phosphate buffer. Under these conditions polymerization occurs at much lower concentrations (ca. 5 mg/ml), which is advantageous when site-directed mutants are being used because only small quantities of the mutants are available. We have characterized the structure of HbS polymers formed in 1.5 M phosphate to determine how their structures are related to the polymers formed under more physiological conditions. Under both sets of conditions fibers are the first species to form. At pHs between 6.7 and 7.3 fibers initially form bundles and then crystals. At lower pHs fibers form macrofibers and then crystals. Fourier transforms of micrographs of the polymers formed in 1.5 M phosphate display the 32- and 64-A(-1) periodicity characteristic of fibers formed in 0.05 M phosphate buffer. The 64-A(-1) layer line is less prominent in Fourier transforms of negatively stained fibers formed in 1.5 M phosphate possibly because salt interferes with staining of the fibers. However, micrographs and Fourier transforms of frozen hydrated fibers formed in high and low phosphate display the same periodicities. Under both sets of reaction conditions HbS polymers form crystals with the same unit cell parameters as Wishner-Love crystals (a = 64 A, b = 185 A, c = 53 A). Some of the polymerization intermediates were examined in the frozen-hydrated state in order to determine whether their structures were significantly perturbed by negative staining. We have also carried out reconstructions of the frozen-hydrated fibers in high and low phosphate to compare their molecular coordinates. The helical projection of the reconstructions in low phosphate shows the expected 14-strand structure. In high phosphate the 14-strand fibers are also formed and their molecular coordinates are the same (within experimental error) as those of fibers formed in 0.05 M phosphate. In addition, the reconstructions of high-phosphate fibers reveal a new minor variant of fiber containing 10 strands. The polymerization products in 1.5 M phosphate buffer were generally indistinguishable from those formed in 0.05 M phosphate buffer. Micrographs of frozen hydrated specimens have facilitated the interpretation of previously published micrographs using negative staining.

Biopolymers↗
PubMed2000

Analysis of gene-specific DNA damage and repair using quantitative polymerase chain reaction.

Soon after discovery of the polymerase chain reaction (PCR), various laboratories have attempted to use quantitative PCR (QPCR) to detect DNA damage in specific gene segments. The development of techniques that facilitate long PCR increased the sensitivity of the assay so that biologically relevant doses of DNA-damaging agents could be assessed. QPCR has been used to survey DNA damage induced by different genotoxicants and to establish the repair kinetics of numerous genes. Current work seeks to analyze damage and repair in specific genes from animals exposed to specific DNA-damaging agents such as oxidative stress.

Alkylating Agents↗
PubMed2000

Positional cloning utilizing genomic DNA microarrays: the Niemann-Pick type C gene as a model system.

A major obstacle in positional cloning is identifying the specific mutated gene from within a large physical contig. Here we describe the application of DNA microarray technology to a defined genomic region (physical map) to identify: (i) exons without a priori sequence data and (ii) the disease gene based on differential gene expression in a recessive disorder. The feasibility was tested using resources from the positional cloning of the Neimann-Pick Type C (NP-C) disease gene, NPC1. To identify NPC1 exons and optimize the technology, an array was generated from genomic fragments of the 110-kb bacterial artificial chromosome, 108N2, which encodes NPC1. First, as a test case for blindly identifying exons, fluorescently labeled NPC1 cDNA identified 108N2 fragments that contained NPC1 exons, many of which also contained intronic sequences and could be used to determine part of the NPC1 genomic structure. Second, to demonstrate that the NPC1 disease gene could be identified based upon differential gene expression, subarrays of 108N2 fragments were hybridized with fluorescently labeled cDNA probes generated from total RNA from hamster cell lines differentially expressing NPC1. A probe derived from the NP-C cell line CT60 did not detect NPC1 exons or other genomic fragments from 108N2. In contrast, several NPC1 exons were detected by a probe generated from the non-NP-C cell line 911D5A13, which was derived from CT60, and expressed NPC1 as a consequence of stable transduction with a YAC that contains NPC1 and encompasses 108N2. Thus, the array technology identified NPC1 as a candidate gene based on a physical contig and differential NPC1 expression between NP-C and non-NP-C cells. This technique should facilitate gene identification when a physical contig exists for a region of interest and mutations result in changes in the mRNA level of the disease gene or portions thereof.

Animals↗
PubMed2000

Expression and purification of imidazole glycerol phosphate synthase from Saccharomyces cerevisiae.

Imidazole glycerol phosphate (IGP) synthase is a glutamine amidotransferase that catalyzes the formation of IGP and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) from N(1)-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-car boxamide ribonucleotide (PRFAR). This enzyme represents a junction between histidine biosynthesis and de novo purine biosynthesis. The recent characterization of the HIS7 gene in the yeast Saccharomyces cerevisiae IGP synthase established that this protein is bifunctional, representing a fusion between the N-terminal HisH domain and a C-terminal HisF domain. Catalytically active yeast HIS7 was expressed in a bacterial system under the control of T7 polymerase promoter. The recombinant enzyme was purified to homogeneity and the native molecular weight and steady-state kinetic constants were determined. The yeast enzyme is distinguished from the Escherichia coli IGP synthase in its utilization of ammonia as a substrate. HIS7 displays a higher K(m) for glutamine and a lower turnover in the ammonia-dependent IGP synthase activity. As observed with the E. coli IGP synthase, HIS7 shows a low basal level glutaminase activity that can be enhanced 1000-fold in the presence of a nucleotide substrate or analog. The purification and characterization of the S. cerevisiae enzyme will enable a more detailed investigation of the biochemical mechanisms that mediate the ammonia-transfer process. The fused structural feature of the HIS7 protein and the development of a high-level production system for the active enzyme elevate the potential for determination of its three-dimensional structure through X-ray crystallography.

Aminohydrolases↗
PubMed2000

Purification and characterization of human alpha-galactosidase A expressed in insect cells using a baculovirus vector.

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. The enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. To perform more extensive biochemical characterization and to develop new approaches for enzyme therapy, a method of producing and purifying recombinant alpha-galactosidase A suitable for scale-up manufacture for use in humans is needed. Previously, a catalytically active recombinant human alpha-galactosidase A was expressed using a baculovirus vector and purified using conventional chromatography. However, the level of expression was too low to permit economical production and the chromatographic techniques used for enzyme purification were not suitable for enzyme to be used in humans. Therefore, the cDNA of the enzyme was cloned to an improved baculovirus vector and the enzyme was expressed in a 15-liter bioreactor using optimized growth conditions. Infection of insect cells by the baculovirus resulted in a significant fivefold increase in the level of secreted recombinant alpha-galactosidase A activity that is compatible with economic manufacturing. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (Poros 20-CM, Poros 20-HQ) and hydrophobic chromatography (Toso-ether, Toso-butyl) using the BioCAD HPLC workstation. These chromatographic steps are readily scalable to larger volumes and are appropriate for the purification of the recombinant human alpha-galactosidase A to be used in clinical trials of enzyme replacement therapy for Fabry disease patients.

Amino Acid Sequence↗
PubMed2000

Glycosylation of prourokinase produced by Pichia pastoris impairs enzymatic activity but not secretion.

Both glycosylated and nonglycosylated forms of recombinant human prourokinase were produced to the level of 20 mg/L by yeast Pichia pastoris in BMMY medium after 2 days of culture. The expressed pro-UK was 98% secreted into the culture medium and easily purified by carboxymethyl cellulose chromatography. More than 99% of pro-UK in the culture medium was found in single-chain form. This was contradictory to a previous finding which found that glycosylation of pro-UK by yeast inhibited its secretion. The absence of glycosylation at Asn302 of pro-UK has no measurable effect on its secretion from the yeast cells. However, the nonglycosylated pro-UK was much less stable in the culture medium, probably due to proteolysis. Nonglycosylated pro-UK from yeast had a clot lysing activity comparable to that of Escherichia coli-derived or mammalian cell-derived recombinant pro-UK. By contrast, the glycosylated yeast pro-UK was less activatable by plasmin and had a lower enzymatic activity against plasminogen and a lower clot lysing activity than nonglycosylated pro-UK from yeast, while their amidolytic activity against S2444 was equivalent. It was concluded that glycosylation of pro-UK by yeast P. pastoris interferes with the catalytic site but not secretion of this protein.

Binding Sites↗
PubMed2000

Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A produced in Pichia pastoris.

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. This enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. An improved method of production of recombinant alpha-galactosidase A for use in humans is needed in order to develop new approaches for enzyme therapy. Human alpha-galactosidase A for use in enzyme therapy has previously been obtained from human sources and from recombinant clones derived from human cells, CHO cells, and insect cells. In this report we describe the construction of clones of the methylotrophic yeast Pichia pastoris that produce recombinant human alpha-galactosidase A. Recombinant human alpha-galactosidase A is secreted by these Pichia clones and the level of production is more than 30-fold greater than that of previously used methods. Production was optimized using variations in temperature, pH, cDNA copy number, and other variables using shake flasks and a bioreactor. Expression of the human enzyme increased with increasing cDNA copy number at 25 degrees C, but not at the standard growth temperature of 30 degrees C. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (POROS 20 CM, POROS 20 HQ) and hydrophobic (Toso-ether, Toso-butyl) chromatography with a BioCAD HPLC Workstation. Purified recombinant alpha-galactosidase A was taken up by fibroblasts derived from Fabry disease patients and normal enzyme levels could be restored under these conditions. Analysis of the carbohydrate present on the recombinant enzyme indicated the predominant presence of N-linked high-mannose structures rather than complex carbohydrates.

Bioreactors↗
PubMed2000

Endothelin-1 and nitric oxide affect human cerebromicrovascular endothelial responses and signal transduction.

Endothelium plays a central role in regulating the vascular tone, blood flow and blood brain barrier (BBB) permeability. The experiments presented here examine the mechanisms by which nitric oxide (NO) and endothelin-1 (ET-1) may be involved in these processes. The findings indicate that ET-1-stimulated [Ca2+]i accumulation occurs through activation of ETA receptor. The capacity of NO to affect this response was indicated by results showing: 1) a two-fold increase in ET-1-stimulated [Ca2+]i by L-NAME, the inhibitor of nitric oxide synthase, and 2) a dose-dependent decrease in [Ca2+]i accumulation by pretreatment with Nor-1 (NO donor). Abrogation of this Nor-1 effect by ODQ (an inhibitor of guanylyl cyclase) or Rp-8-pCPT-cGMPS (an inhibitor of protein kinase G) and inhibition of ET-1 stimulated intracellular Ca2+ accumulation by 8-bromo-cGMP (a permeable, analog of cGMP) substantiate the involvement of interplay between ET-1 and NO in [Ca2+]i accumulation in HBMEC. ET-1 treatment also increased thickness of F-actin cytoskeletal filaments in HBMEC. This effect was attenuated by pretreatment with NO; NO also rarefied F-actin filaments in control cultures. The findings support a linkage between NO and ET-1 in regulating microvascular tone, microcirculation and BBB permeability and indicate a role for cGMP/cGMP protein kinase system and cytoskeletal changes in responses of HBMEC.

Actins↗
PubMed2000

Study on P16 gene in acute leukemia.

To study the change of suppressing cancer gene P16 in acute leukemia, the P16 antigen expression of leukemia cell surfaces in 61 cases were investigated with ABC assay and gene structural defects in 51 cases of acute leukemia were examined with multiple comparative PCR method. It was found that antigen expression of P16 in leukemia was obviously lower than that innormal subjects (P < 0.001). At the same time, antigen expression in All was lower than that AML (P < 0.05). No significant difference was found between the complete remission (CR) and non-remission (NR) subjects from AML and ALL groups (P > 0.05). THe exon 2 of P16 gene showed homozygous deletion only inn 4 cases out of 30 cases in ALL. No structural defect was revealed in 21 cases of AML. It was suggested that expression defect of P16 gene was a main cause in development and progression of acute leukemia, and structural defect of exon 2 was not a primary molecular event.

Adolescent↗
PubMed2000

The influence of curcumin on the cell cycle of HL-60 cells and contrast study.

The mechanisms of curcumin inhibiting the proliferation of HL-60 cells were investigated. Acute myeloid leukemic cell line HL-60 was studied by using cell culture, NBT reduction, SABC method measuring BrdU incorporation rate, FCM measuring DNA contents and TUNEL method determining apoptotic cell percentage. Curcumin inhibited proliferation of HL-60 cells in a dose- and time-dependent manner. When the HL-60 cells were treated with 25 mumol/L curcumin for 48 h, the inhibitory rate was 60.71% +/- 1.20%. The study on BrdU incorporation rate and the distribution of DNA content and NBT reduction indicated that curcumin arrested the cells in G2/M phase of cell cycle at first, then in G0/G1 phase, the whole cell cycle progression was slowed down and DNA synthesis activities was halted. The arrested cells went to apoptosis instead of differentiation. The comparative study indicated that the ability of curcumin regulating the cell cycle of HL-60 cells was stronger than As2O3 and the acting points of curcumin were not completely consistent with those of VP-16. It was suggested that curcumin was able to regulate, to some extent, the G1/S and G2/M transmit checkpoints and disturb the HL-60 cell cycle to induce apoptosis.

Antineoplastic Agents↗
PubMed2000

Preliminary study of stereotactic radiotherapy for lung cancer.

From March 1997 to November 1999, 45 patients with lung cancer were treated by a stereotactic radiotherapy, with 15 cases treated by a stereotactic radiotherapy alone, and 30 cases by the external radiotherapy plus stereotactic radiotherapy. The clinical target volume was 1.89-187.26 cm3 with the median being 18.17 cm3. The doses of plan target volume (PTV) edge was 16-30 Gy/2-3 times and the doses of center was 120% to 150% of PTV edge doses. The overall response rate was 84.4% (38/45), with 11 complete response (CR) and 27 partial response (PR). This study confirmed that the stereotactic radiotherapy is a safe and effective therapy for lung cancer. For those early-stage patients who can tolerate neither operation nor even conventional radiotherapy for various reasons, it can both achieve therapeutic purpose and improve quality of life.

Adenocarcinoma↗
PubMed2000

Alterations in the expression and activity of creatine kinase-M and mitochondrial creatine kinase subunits in skeletal muscle following prolonged intense exercise in rats.

Creatine kinase (CK) isoenzymes are important structural and energy metabolism components in skeletal muscle. In this study, CK isoenzyme alterations were examined in male rats, with an 8% body mass weight attached to their tail. The rats were either forced to swim for 5 h (5S, n = 51), or were pre-trained for 8 days and then forced to swim for 5 h (T5S, n = 48). Rats were sacrificed either immediately (0 h PS), 3 h (3 h PS), or 48 h post-swimming (48 h PS). Serum CK was increased significantly (P < 0.01) 6.2- and 2.0-fold at 0 h PS following the 5S and T5S protocols, respectively. However, training (T5S protocol) significantly (P < 0.01) decreased CK release. Soleus and white gastrocnemius (WG) CK activity was significantly decreased following the 5S protocol (P < 0.05), but not following the T5S protocol. The CK-M activity of the soleus muscle was significantly (P < 0.05) decreased at 0 h PS following both the 5S and T5S protocols, and returned to control values at 3 h PS. The CK-M activity of the WG was significantly (P < 0.05) decreased at 0 h PS following the 5S protocol. Sarcomeric mitochondrial CK (sCK-Mit) was decreased significantly (P < 0.01) at 0 h PS (20%), 3 h PS (14%), 24 h PS (22%), and 48 h PS (15%) following the 5S protocol. However, sCK-Mit was decreased significantly (P < 0.01) only at 0 h PS (7%) following the T5S. The results of this study demonstrate that prolonged intense exercise causes a loss of skeletal muscle CK-M and sCK-Mit activity and that training prior to the prolonged intense exercise attenuates the exercise-induced CK-M and sCK-Mit loss in both red and white skeletal muscles.

Animals↗
PubMed2000

Antisense prevention of neuronal damages following head injury in mice.

Closed head injury (CHI) is an important cause of death among young adults and a prominent risk factor for nonfamilial Alzheimer's disease. Emergency intervention following CHI should therefore strive to improve survival, promote recovery, and prevent delayed neuropathologies. We employed high-resolution nonradioactive in situ hybridization to determine whether a single intracerebro-ventricular injection of 500 ng 2'-O-methyl RNA-capped antisense oligonucleotide (AS-ODN) against acetylcholinesterase (AChE) mRNA blocks overexpression of the stress-related readthrough AChE (AChE-R) mRNA splicing variant in head-injured mice. Silver-based Golgi staining revealed pronounced dendrite outgrowth in somatosensory cortex of traumatized mice 14 days postinjury that was associated with sites of AChE-R mRNA overexpression and suppressed by anti-AChE AS-ODNs. Furthermore, antisense treatment reduced the number of dead CA3 hippocampal neurons in injured mice, and facilitated neurological recovery as determined by performance in tests of neuromotor coordination. In trauma-sensitive transgenic mice overproducing AChE, antisense treatment reduced mortality from 50% to 20%, similar to that displayed by head-injured control mice. These findings demonstrate the potential of antisense therapeutics in treating acute injury, and suggest antisense prevention of AChE-R overproduction to mitigate the detrimental consequences of various traumatic brain insults.

Acetylcholinesterase↗
PubMed2000

Determination of marker constituents in radix Glycyrrhizae and radix Notoginseng by near infrared spectroscopy.

High-performance liquid chromatographic (HPLC) methods were developed for the determination of glycyrrhizin in radix Glycyrrhizae and ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf and Rg, in radix Notoginseng. These methods were used as reference methods for near-infrared (NIR) spectroscopy. Spectroscopic calibrations were developed for the determination of glycyrrhizin, the total content of ginsenosides and the individual major ginsenosides Rb1, Rd, Re and Rg1. Standard errors of cross validation (SECV) were 1.22 mg g(-1) for glycyrrhizin (concentration range 21.3-34.1 mg g(-1)) and 0.99 mg g(-1) for the sum of ginsenosides (concentration range 55.3-71.1 mg g(-1)). The corresponding coefficients of determination (R2) were 0.94 and 0.98, respectively. The SECVs were generally less than a factor of 2.5 of the repeatability standard deviation of the HPLC methods.

Calibration↗
PubMed2000

Effect of retinoic acid and its complexes with transition metals on human bladder cancer cell line EJ in vitro.

The aim of this study was to investigate the effect of retinoic acid (RA) and its complexes with transition metals on the bladder cancer cell line EJ. Retinoic acid complexes with transition metals Cu, Co, Zn, and Ni were prepared. Cell proliferation was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in the presence of RA or its complexes with transition metals Cu, Co, Zn, and Ni ¿Cu(RA)2.3H2O, Co(RA)2.3H2O, Zn(RA)2.4H2O, and Ni(RA)2.3H2O¿. Colony formation in soft agar culture, A agglutination reaction, and lactic acid dehydrogenase isoenzyme assay were performed in the cells treated with these drugs to estimate the induced differentiation. p53 or c-Ha-ras expression in drug-treated cells was assayed by ABC immunocytochemistry technique. The results demonstrate that EJ cells treated with the drugs become less confluent and tend to exhibit normal characteristics. Although RA and its complexes showed inhibition to proliferation of EJ cells at the concentrations of 10(-6) mmol/l, the inhibition induced by Ni(RA)2.3H2O was much more marked than that by RA. EJ cells were growth inhibited by RA or Ni(RA)2.3H2O from 48 to 96 h at the concentration of 10(-8) mol/l. The levels of LDH4 and LDH5 in the cells were greatly increased by RA. Nevertheless, Ni(RA)2.3H2O did not affect LDH isoenzyme in EJ cells. The number of colony formations of EJ cells in soft agar culture was decreased by RA or Ni(RA)2.3H2O. The percentage of colony formation in soft agar culture was much lower in EJ cells treated with Ni(RA)2.3H2O than with RA. The required concentration of A agglutination reaction was more increased for EJ cells treated with RA or Ni(RA)2.3H2O than for the control and was further increased in cells treated with Ni(RA)2.3H2O. Mutant p53 expression was more decreased in the EJ cells treated with RA or Ni(RA)2.3H2O than in the control. Although RA at the concentration of 10(-6) mmol/l caused lower p21 expression, Ni(RA)2.3H2O did not affect p21 expression in EJ cells. Therefore, RA and its transition metal complexes have a potential use in the treatment of bladder cancer.

Agar↗