Biomedical subjects

F Chen

Publications and source records attributed to F Chen.

At least 19 recordsLinked to original sources

PubMed2003

Identification of bulb from Fritillaria cirrhosa by PCR with specific primers.

Bulb of Fritillaria cirrhosa is an important traditional Chinese herbal medicine. According to the Chinese Pharmacopoeia (1995), it is commonly used as an antitussive and expectorant. Many young bulbs from species of Fritillaria are similar to those of F. cirrhosa, but they are different in price and quality. Therefore, there are many young bulbs from species of Fritillaria that could fake those of F. cirrhosa on the commercial market. The coding region of 5S-rRNA is highly conserved in higher eukaryotes. The 5S-rRNA spacer region sequences of F. thunbergii, F. pallidiflora, F. ussuriensi, F. delavayi, F.cirrhosa, F. anhuiensis, F. puqiensis were cloned by PCR with a pair of primers located within the conserved coding region. Based on sequences analyses of the 5S-rRNA spacer region from the 7 species, a specific sequence was found in F. cirrhosa. A pair of specific primers was designed for differentiating the bulbs of F. cirrhosa from each other by PCR. This result indicated that the method is rapid, more accurate and applicable in identification of the bulbs of F. cirrhosa at the DNA level.

Base Sequence↗

PubMed2002

Demonstration of the lateral casimir force.

The lateral Casimir force between a sinusoidally corrugated gold coated plate and large sphere was measured for surface separations between 0.2 to 0.3 microm using an atomic force microscope. The measured force shows the required periodicity corresponding to the corrugations. It also exhibits the necessary inverse fourth power distance dependence. The obtained results are shown to be in good agreement with a complete theory taking into account the imperfectness of the boundary metal. This demonstration opens new opportunities for the use of the Casimir effect for lateral translation in microelectromechanical systems.

Journal Article↗

PubMed2002

Hyaluronic acid and chondroitin sulphate A rapidly promote differentiation of immature DC with upregulation of costimulatory and antigen-presenting molecules, and enhancement of NF-kappaB and protein kinase activity.

Dendritic cells (DCs) have been identified as effective antigen-presenting cells (APCs). We demonstrate that extracellular matrix (ECM), hyaluronic acid (HA) and chondroitin sulphate A (CSA), in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), can rapidly promote the differentiation of monocyte-derived immature DCs, as characterized by the remarkable upregulation of human leucocyte antigen (HLA-DR), CD40, CD54, CD80 and CD86 expression to levels higher than those in the DCs generated by culturing with GM-CSF and interleukin (IL)-4 for 7 days and aggregation of the cells within 48 h. The upregulation of expression of HLA-DR, CD40, CD54, CD80 and CD86 was dose-dependent. Further studies showed that HA and CSA were able to augment nuclear factor (NF)-kappaB activity, as determined by gel mobility shift assay and promote protein phosphorylation. Inhibition of NF-kappaB by pyrolidine dithiocarbamate and sodium salicylate, and serine-threonine and tyrosine kinase by starosporine as well as phosphatidylinositide-3-kinase (PI-3-K) by wortmannin could prevent the effects of HA and CSA on the expression of HLA-DR, CD40, CD80 and CD86 in various degrees. Thus, our data demonstrate that HA or CSA can effectively and rapidly promote the differentiation of immature DC, suggesting that HA and CSA may possess a potential capacity in regulating immune responses.

Antigen Presentation↗

PubMed2002

[Surgical treatment of Marfan syndrome; analysis of the patients required multiple surgical interventions].

Without treatment, the life expectancy of patients with Marfan syndrome is reduced by the associated cardiovascular abnormalities. In this study, we reviewed our experience of the patients with Marfan syndrome who required multiple surgical interventions to identify the optimal treatment for these patients. Between January 1986 and December 2000, 44 patients with Marfan syndrome were operated on at Shizuoka City Hospital (SCH). Among them, 10 patients (22.7%) underwent multiple surgical interventions. There were 5 male and 5 female patients with a mean age of 40.6 +/- 16.1 years at the initial surgery. Only one patient was operated on at another hospital for his first, second, and third operations. His fourth operation was carried out at SCH. The remaining 9 patients underwent a total of 14 additional surgical procedures at SCH. Computed tomography (CT) scans were taken every 6 months postoperatively, and aortic diameter greater than 60 mm was considered as the indication for the additional surgery. There were no early death and one late death. The causes of additional surgery were enlargement of true aneurysm in 6, enlargement of residual dissection in 4, new dissection in 4, false aneurysm at the coronary anastomosis of Bentall procedure in 1. In 9 patients, both ascending and descending aorta were replaced. Among these 9 patients, only 3 patients underwent total arch replacement, and remaining 6 patients had their arch left in place with or without dissection. Our current strategy of the treatment of Marfan patients with acute type A dissection is total arch replacement with an elephant trunk at the initial emergent surgery.

Adolescent↗

PubMed2001

An anion-induced regio- and chemoselective acylation and its application to the synthesis of an anticancer agent.

[reaction--see text] An efficient Grignard- and organolithium-induced regio- and chemoselective anionic acylation is reported. A number of tricyclic ketones are prepared in good to excellent yields via this method. This method is complementary to the Frieldel-Crafts acylation for electron-deficient substrates. A novel anisole-based Grignard reagent was developed to effect the cyclization of sterically hindered substrates. This novel reagent has been successfully applied to the synthesis of Sch 66336, a candidate for oncologic treatment.

Acylation↗

PubMed2001

Application of statistically-based experimental designs for the optimization of eicosapentaenoic acid production by the diatom Nitzschia laevis.

Statistically based experimental designs were applied to the optimization of medium components and environmental factors for eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis in heterotrophic conditions. First, the Plackett-Burman design was used to evaluate the effects of variables including medium components and environmental factors on cell growth and EPA production. Among these variables, NaCl, CaCl(2), PI metal solution, pH, and temperature were identified to have the significant effects (with confidence level > 90 %). Subsequently, the concentrations of NaCl, CaCl(2), PI metal solution as well as the values of pH and temperature were optimized using central composite design. The cell growth and EPA production were found to respectively correlate to NaCl, CaCl(2), pH, and temperature that could be represented by second-order polynomial models. The optimal values of the four parameters were determined by response surface and numerical analyses as 8 g/L NaCl, 0.10 g/L CaCl(2), pH 8.5 and 19.8 degrees C for cell dry weight (DW), and 14 g/L NaCl, 0.10 g/L CaCl(2), pH 8.5 and 18 degrees C for EPA production, respectively. The subsequent verification experiments confirmed the validity of the models. This optimization strategy led to a DW of 9 g/L, an EPA yield of 280 mg/L and an EPA productivity of 28 mg/L/d, respectively, which were considerably higher than those obtained in the previous studies.

Biotin↗

PubMed2001

Preparative isolation and purification of salidroside from the Chinese medicinal plant Rhodiola sachalinensis by high-speed counter-current chromatography.

High-speed counter-current chromatography was applied to the isolation and purification of salidroside from the Chinese medicinal plant Rhodiola sachalinensis A. Bor. The crude salidroside was obtained by extraction with methanol from Rhodiola sachalinensis A. Bor. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-butanol-ethyl acetate-water (2:3:5, v/v) was successfully performed yielding salidroside (32 mg) at 98% purity from 250 mg of the crude extract in a one-step separation.

Chromatography, High Pressure Liquid↗

PubMed2001

Characterization of the DNA-binding property of Smad5.

Activation of TGF-beta superfamily receptors leads to phosphorylation of Smad proteins which function as transcription factors to regulate gene expression. Previous studies have indicated that Smad5, together with Smad1 and Smad8, participates in signaling downstream of BMP receptors. To characterize the DNA-binding characteristics of Smad5, we used the GST-Smad5 N-terminal fusion protein to select for random oligonucleotide sequences that were able to binds the protein. As a result, we found that Smad5 is able to bind a consensus sequence TGTGC. We further used the Smad7 promoter sequence that contains a Smad-binding element (SBE), GTCTAGAC to determine how mutations in each nucleotide in the SBE affects the binding with Smad5, compared with the binding with Smad1, Smad2, Smad3, Smad4, and Smad8. Interestingly, Smad5, but not Smad1 and Smad8, was able to bind the SBE, at a level similar to the binding by Smad3 and Smad4. However, mutations at the SBE had different effect on the binding with Smad5, compared to that with Smad3 and Smad4. These studies suggest that even though Smad5 falls into the same subfamily with Smad1 and Smad8 in mediating the signaling by BMP receptors, it has an unique DNA-binding property that is similar to Smad3, which specifically transduces signaling for TGF-beta and activin receptors.

Animals↗

PubMed2001

EBNA promoter usage in EBV-negative Burkitt lymphoma cell lines converted with a neomycin-resistant EBV strain.

Latent Epstein-Barr virus (EBV) uses two alternative strategies to express the Epstein-Barr nuclear antigens (EBNAs). Resting normal B cells harboring latent virus and Burkitt's lymphoma (BL) cells use monocistronic messages generated from the Q promoter (restricted strategy). EBV-transformed immunoblasts express all EBNAs by using giant messages generated from the W/C promoter (full program). Whether the virus establishes the restricted program on primary infection of a BL cell (or its progenitor) or, alternatively, whether such cells are generated by phenotypic down-regulation from the immunoblast is unclear. We found previously that conversion of EBV-negative BL lines to EBV-positive sublines required repeated exposure to large virus doses. The converted sublines used the full program. However, the possibility that cells with a full program had a selective advantage during the long period of in vitro passage could not be excluded. We therefore infected EBV-negative BL lines with recombinant EBV carrying a neomycin resistance marker. Most convertants of the 12 lines tested were positive for YUK splicing, indicative of the full program, but some were also positive for the restricted QUK splice program. One convertant DG75 line showing both YUK and QUK was cloned and gave rise to stable QUK users. We conclude that EBV infection of established BL lines can give rise to subclones with either the full or the restricted program. The fact that all EBVs carrying BL lines use the restricted program in vitro may be a consequence of immunoselection.

Anti-Bacterial Agents↗

PubMed2001

Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils.

beta-Amyloid plaques and neurofibrillary tangles (NFTs) are the defining neuropathological hallmarks of Alzheimer's disease, but their pathophysiological relation is unclear. Injection of beta-amyloid Abeta42 fibrils into the brains of P301L mutant tau transgenic mice caused fivefold increases in the numbers of NFTs in cell bodies within the amygdala from where neurons project to the injection sites. Gallyas silver impregnation identified NFTs that contained tau phosphorylated at serine 212/threonine 214 and serine 422. NFTs were composed of twisted filaments and occurred in 6-month-old mice as early as 18 days after Abeta42 injections. Our data support the hypothesis that Abeta42 fibrils can accelerate NFT formation in vivo.

Aged↗

PubMed2001

The role of AP-1 in the transcriptional regulation of the rat apical sodium-dependent bile acid transporter.

Ileal reclamation of bile salts, a critical determinant of their enterohepatic circulation, is mediated primarily by the apical sodium-dependent bile acid transporter (ASBT=SLC10A2). We have defined mechanisms involved in the transcriptional regulation of ASBT. The ASBT gene extends over 17 kilobases and contains five introns. Primer extension analysis localized two transcription initiation sites 323 and 255 base pairs upstream of the initiator methionine. Strong promoter activity is imparted by both a 2.7- and 0.2-kilobase 5'-flanking region of ASBT. The promoter activity is cell line specific (Caco-2, not Hep-G2, HeLa-S3, or Madin-Darby canine kidney cells). Four distinct specific binding proteins were identified by gel shift and cross-linking studies using Caco-2 or rat ileal nuclear extracts. Two AP-1 consensus sites were identified in the proximal promoter. DNA binding and promoter activity could be abrogated by mutation of the proximal AP-1 site. Supershift analysis revealed binding of c-Jun and c-Fos to this AP-1 element. Co-expression of c-Jun enhanced promoter activity in Caco-2 cells and activated the promoter in Madin-Darby canine kidney cells. Region and developmental stage-specific expression of ASBT in the rat intestine correlated with the presence of one of these DNA-protein complexes and both c-Fos and c-Jun proteins. A specific AP-1 element regulates transcription of the rat ASBT gene.

Animals↗

PubMed2001

Preparative isolation and purification of astaxanthin from the microalga Chlorococcum sp. by high-speed counter-current chromatography.

High-speed counter-current chromatography was applied to the isolation and purification of astaxanthin from microalgae. The crude astaxanthin was obtained by extraction with organic solvents after the astaxanthin esters were saponified. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (5:5:6.5:3, v/v) was successfully performed yielding astaxanthin at 97% purity from 250 mg of the crude extract in a one-step separation.

Chlorophyta↗

PubMed2001

Preparative isolation and purification of six diterpenoids from the Chinese medicinal plant Salvia miltiorrhiza by high-speed counter-current chromatography.

A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of six diterpenoids. dihydrotanshinone I, cryptotanshinone, methylenetanshiquinone, tanshinone I, tanshinone IIA and danshenxinkun B from the Chinese medicinal plant Salvia miltiorrhiza Bunge. The crude diterpenoids were obtained by extraction with ethanol-n-hexane (1:1, v/v) from S. miltiorrhiza Bunge. Preparative HSCCC with the two-phase solvent systems A composed of n-hexane-ethanol-water (10:5.5:4.5, v/v) and B composed of n-hexane-ethanol-water (10:7:3, v/v) was successfully performed in a stepwise elution yielding six relatively pure diterpenoids from 300 mg of the crude extract in a single run. The purities of dihydrotanshinone I, cryptotanshinone, methylenetanshiquinone, tanshinone I, tanshinone IIA and danshenxinkun B were 88.1, 98.8, 97.6, 93.5, 96.8 and 94.3%, respectively.

Chromatography, High Pressure Liquid↗

PubMed2001

RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples.

The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNAlater, and held in cold storage for 3 months was compared. Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated that the cell storage treatments tested were equally effective at maintaining their total RNA content. There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage. Intact mRNA transcripts of the RuBisCO (rbcL) and nir genes were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage. In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. In addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples.

Bacteria↗

PubMed2001

Production and characterization of an antiserum which recognizes the native receptor for thyrotropin-releasing hormone.

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa and a minor broad band at 100-120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy.

Animals↗

PubMed2001

The three-dimensional structure of the C-terminal DNA-binding domain of human Ku70.

The proteins Ku70 (69.8 kDa) and Ku80 (82.7 kDa) form a heterodimeric complex that is an essential component of the nonhomologous end joining DNA double-strand break repair pathway in mammalian cells. Interaction of Ku with DNA is central for the functions of Ku. Ku70, which is mainly responsible for the DNA binding activity of the Ku heterodimer, contains two DNA-binding domains. We have solved the solution structure of the Ku80-independent DNA-binding domain of Ku70 encompassing residues 536-609 using nuclear magnetic resonance spectroscopy. Residues 536-560 are highly flexible and have a random structure but form specific interactions with DNA. Residues 561-609 of Ku70 form a well defined structure with 3 alpha-helices and also interact with DNA. The three-dimensional structure indicates that all conserved hydrophobic residues are in the hydrophobic core and therefore may be important for structural integrity. Most of the conserved positively charged residues are likely to be critical for DNA recognition. The C-terminal DNA-binding domain of Ku70 contains a helix-extended strand-helix motif, which occurs in other nucleic acid-binding proteins and may represent a common nucleic acid binding motif.

Amino Acid Sequence↗

PubMed2001

Signals transduced by Ca(2+)/calcineurin and NFATc3/c4 pattern the developing vasculature.

Vascular development requires an orderly exchange of signals between growing vessels and their supporting tissues, but little is known of the intracellular signaling pathways underlying this communication. We find that mice with disruptions of both NFATc4 and the related NFATc3 genes die around E11 with generalized defects in vessel assembly as well as excessive and disorganized growth of vessels into the neural tube and somites. Since calcineurin is thought to control nuclear localization of NFATc proteins, we introduced a mutation into the calcineurin B gene that prevents phosphatase activation by Ca(2+) signals. These CnB mutant mice exhibit vascular developmental abnormalities similar to the NFATc3/c4 null mice. We show that calcineurin function is transiently required between E7.5 and E8.5. Hence, early calcineurin/NFAT signaling initiates the later cross-talk between vessels and surrounding tissues that pattern the vasculature.

Active Transport, Cell Nucleus↗

PubMed2001

An enzymatic assay for quantifying sphingomyelin in tissues and plasma from humans and mice with Niemann-Pick disease.

Sphingomyelin is an important lipid component of cell membranes and lipoproteins which can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase, and the resultant accumulation of sphingomyelin in cells and tissues. In this paper we report a new, enzyme-based method to quantify the levels of sphingomyelin in tissues and plasma of normal individuals and NPD patients. The method utilizes sphingomyelinase from Bacillus cereus to completely hydrolyze the sphingomyelin into ceramide. Quantification of the sphingomyelin-derived ceramide is accomplished using Escherichia coli diacylglycerol (DAG) kinase and [gamma-(32)P]ATP. The resulting [(32)P]ceramide is quantified using a phosphor-imager system following TLC separation. This procedure allowed quantification of sphingomyelin over a broad range from 10 pmol to 1 nmol. To validate this assay we quantified sphingomyelin in plasma and tissues obtained from normal and NPD mice and humans. The sphingomyelin content in adult homozygous (-/-) or heterozygous (+/-) NPD mouse plasma was significantly elevated compared to that of normal mice (up to twofold). Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 40 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several type B NPD patients also were significantly elevated compared to normal individuals of the same age. Based on these results we propose that this new, enzyme-based procedure can provide sensitive and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could be a useful tool for the diagnosis of NPD and the evaluation of NPD treatment protocols, as well as for the study of ceramide-mediated apoptosis since the method provides the simultaneous determination of sphingomyelin and ceramide in the same lipid extract.

Animals↗