Biomedical subjects

F Chen

Publications and source records attributed to F Chen.

At least 37 records · Page 2Linked to original sources

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↗

PubMed2001

Expression of the neuronal cyclin-dependent kinase 5 activator p35Nck5a in human monocytic cells is associated with differentiation.

Although cyclin-dependent kinase 5 (Cdk5) is widely expressed in human tissues, its activator p35Nck5a is generally considered to be neuron specific. In addition to neuronal cells, active Cdk5 complexes have been reported in developing tissues, such as the embryonic muscle and ocular lens, and in human leukemia HL60 cells induced to differentiate by an exposure to 1,25-dihydroxyvitamin D(3); however, its activator in these cells has not been demonstrated. The results of this study indicate that p35Nck5a is associated with Cdk5 in monocytic differentiation of hematopoietic cells. Specifically, p35Nck5a is expressed in normal human monocytes and in leukemic cells induced to differentiate toward the monocytic lineage, but not in lymphocytes or cells induced to granulocytic differentiation by retinoic acid. It is present in a complex with Cdk5 that has protein kinase activity, and when ectopically expressed together with Cdk5 in undifferentiated HL60 cells, it induces the expression of CD14 and "nonspecific" esterase, markers of monocytic phenotype. These observations not only indicate a functional relationship between Cdk5 and p35Nck5a, but also support a role for this complex in monocytic differentiation. (Blood. 2001;97:3763-3767)

Cell Cycle Proteins↗

PubMed2001

Contrasting roles of NF-kappaB and JNK in arsenite-induced p53-independent expression of GADD45alpha.

Growth arrest and DNA damage-inducible protein 45alpha (GADD45alpha) is an important cell cycle checkpoint protein that arrests cells at G2/M phase by inhibiting the activity of G2-specific kinase, cyclin B/p34cdc2. We report here that arsenite induces GADD45alpha expression in a p53-independent fashion and that this GADD45alpha induction by arsenite is regulated by NF-kappaB and c-Jun-N-terminal kinase (JNK) oppositely. In human bronchial epithelial cells overexpressing a kinase-mutated form of IkappaB kinase beta (IKKbeta-KM), the activation of NF-kappaB was inhibited. However, the G2/M cell cycle arrest and expression of GADD45alpha was substantially enhanced in response to arsenite in these cells. Expression of a dominant-negative mutant of SEK1 that blocks JNK activation decreased arsenite-induced GADD45alpha expression. Analysis of GADD45alpha expression in both wild-type and p53-/- fibroblasts indicated that the induction of GADD45alpha by arsenite was independent of the status of p53 protein.

Arsenites↗

PubMed2001

Characterization of the murine Lbx2 promoter, identification of the human homologue, and evaluation as a candidate for Alström syndrome.

The murine Lbx2 gene is a member of the ladybird family of homeobox genes, which is expressed in the developing urogenital system, eye, and brain. Using transgenic mice, we demonstrate that 9 kb of the 5' flanking region of mouse Lbx2 is able to direct expression of a reporter gene in a tissue-specific manner recapitulating the endogenous expression pattern. This regulatory region provides a novel reagent allowing for transgenic expression in the developing urogenital ridge. In addition, we describe the identification of the human homologue, LBX2. Comparison of the human LBX2 and mouse Lbx2 sequences upstream of the coding regions reveals sequence conservation suggesting conserved regulatory regions. Both the human LBX2 and the mouse Lbx2 genes have similar genomic structures and are composed of two exons separated by an intron. We mapped the mouse Lbx2 gene to 35 cM on chromosome 6 and the human LBX2 gene to a homologous region of chromosome 2p13. This is a candidate region for several inherited disorders, including Alström syndrome, a disorder that includes ocular, urogenital, and renal abnormalities. Given the expression pattern of Lbx2, the chromosomal location in humans, and the potential function of mammalian ladybird genes, we have begun to analyze patients with ocular disorders and those with Alström syndrome for mutations in LBX2. Although polymorphisms were identified, our results indicate that mutations in the coding region of LBX2 do not account for Alström syndrome in the six kindreds analyzed.

Amino Acid Sequence↗

PubMed2001

Determination of iodide in seawater and urine by size exclusion chromatography with iodine-starch complex.

A novel method for the determination of iodide by size exclusion chromatography was established. The method was simple and highly sensitive with good precision. Iodide was converted to iodine, then sequestered with starch, and separated from the matrix using a Shim-pack DIOL-150 (250 x 7.9 mm) size exclusion column with methanol-0.01 mol l(-1) aqueous phosphoric acid (10:90, v/v) as mobile phase at 1.2 ml min(-1) and UV detection at 224 nm. The calibration graph was linear from 1.0 ng ml(-1) to 100.0 ng ml(-1) for iodide with a correlation coefficient of 0.9992 (n=6). The detection limit was 0.2 ng ml(-1). The method was successfully applied to the determination of iodide in seawater and urine. The recovery was from 92% to 103% and the relative standard deviation was in the range of 1.5% to 3.7%.

Calibration↗

PubMed2001

[Effects of cigarette smoke extract on the expression of beta-catenin and tyrosine phosphorylation in airway epithelial cells].

OBJECTIVE: To explore the possible mechanism of injury by smoking and its repairing, the effects of cigarette smoke extract on the expression of beta-catenin (beta-cat), c-Src and level of phosphoric tyrosine in airway epithelial cells (AEC). METHODS: Pig AECs were cultured in vitro. After CSE and tyrphostin 25 exposure, (1) the expression of beta-cat was determined by immunocytochemical stain in 300 randomly selected AECs of each group, (2) the level of phosphoric tyrosine, content of c-Src and beta-cat mRNA in AECs were evaluated by immunocytochemical stain, in situ hybridization and image analysis in 30 randomly selected AECs of each group. RESULTS: After exposure to CSE for 4 hours and 24 hours, the levels of phosphoric tyrosine were 1.51 and 3.44 times of that of the control groups of the same time (P < 0.0001) respectively; the ratio of positive stain of beta-cat on membrane was markedly lower than that in the control groups (78.3% vs 83.3%, 77.7% vs 81.3%, P < 0.0001), and the cytoplasmic content of c-Src decreased 47.9% and 51.4% (P < 0.0001) compared with the control groups of the same time, respectively. While dealt with CSE and tyrphostin 25 together, the level of phosphoric tyrosine, the expression of beta-cat and c-Src resembled those in normal control. Contends of beta-cat mRNA decreased in the both two situations (P < 0.0010). CONCLUSION: CSE decreased the membranous expression of beta-cat and increased the level of tyrosine phosporylation of AECs. The change of beta-cat expression might mediate the airway epithelial injuries caused by smoking, and tyrphostin 25 might prevent such injuries through inhibiting the tyrosine kinase.

Animals↗

PubMed2001

Additional t(11;17)(q23;q21) in a patient with Philadelphia-positive mixed lineage antigen-expressing leukemia.

We describe very uncommon phenotypic and cytogenetic findings in a 40-year-old female with blast phase of Philadelphia chromosome (Ph)-positive CML. In addition to the t(9;22)(q34;q11) that was detected in all metaphases, a t(11;17)(q23;q21) was identified in 15 of 20 metaphases. Reverse transcription-polymerase chain reaction showed the major and minor bcr/abl fusion transcripts in the cells from a bone marrow (BM) sample. Fluorescence in situ hybridization (FISH) analysis also showed that fusion signals of the bcr and abl probes were found in 95% of blastic cells and in 64% of neutrophils. MLL gene rearrangement was also detected in some blastic cells but not in neutrophils by FISH analysis. Phenotypically, blastic cells expressed mixed lineage antigens such as CD34, CD33, CD13, CD19, CD7, and CD41. Immunogenotypically, some population of BM cells showed monoclonal rearrangements of immunoglobulin heavy chain and T-cell receptor gamma chain genes by Southern blot analysis. Clinical course was aggressive, and therapy was poorly tolerated. Such findings seem to support an association between Ph and an abnormality of 11q23 with poor prognosis, and suggest that the expression of both abnormal genes may be related to this mixed lineage antigen-expressing leukemia.

Adult↗

PubMed2001

The small heat shock protein alpha B-crystallin negatively regulates cytochrome c- and caspase-8-dependent activation of caspase-3 by inhibiting its autoproteolytic maturation.

Caspases are universal effectors of apoptosis. The mitochondrial and death receptor pathways activate distinct apical caspases (caspase-9 and -8, respectively) that converge on the proteolytic activation of the downstream executioner caspase-3. Caspase-9 and -8 cleave procaspase-3 to produce a p24 processing intermediate (composed of its prodomain and large subunit), which then undergoes autoproteolytic cleavage to remove the prodomain from the active protease. Recently, several heat shock proteins have been shown to selectively inhibit the mitochondrial apoptotic pathway by disrupting the activation of caspase-9 downstream of cytochrome c release. We report here that the small heat shock protein alphaB-crystallin inhibits both the mitochondrial and death receptor pathways. In S-100 cytosolic extracts treated with cytochrome c/dATP or caspase-8, alphaB-crystallin inhibits the autoproteolytic maturation of the p24 partially processed caspase-3 intermediate. In contrast, neither the closely related small heat shock protein family member Hsp27 nor Hsp70 inhibited the maturation of the p24 intermediate. We also demonstrate that alphaB-crystallin co-immunoprecipitates with the p24 partially processed caspase-3 in vivo. Taken together, our results demonstrate that alphaB-crystallin is a novel negative regulator of apoptosis that acts distally in the conserved cell death machinery by inhibiting the autocatalytic maturation of caspase-3.

Apoptosis↗

PubMed2001

UV light-induced degradation of RNA polymerase II is dependent on the Cockayne's syndrome A and B proteins but not p53 or MLH1.

It has been hypothesized that the degradation of the largest subunit of RNA polymerase II (polIILS) is required for transcription-coupled repair (TCR) of UV light-induced transcription-blocking lesions. In this study we further investigated the mechanism of UV-induced degradation of polIILS using cell lines with specific defects in TCR or in the recovery of RNA synthesis. It was found that the hypophosphorylated IIa form of polIILS rapidly decreased following UV-irradiation in all cell lines tested. Inhibition of proteasome activity resulted in an increase of the hyperphosphorylated IIo form of polIILS in UV-irradiated cells, while inhibition of CTD-kinases resulted in the retention of the IIa form. In UV-irradiated Cockayne's syndrome cells, which are defective in TCR, the levels of the IIo form increased in a similar manner as when proteasome inhibitors were added to UV-irradiated normal cells. In contrast, TCR-deficient HCT116 cells, which lack the mismatch repair protein MLH1, showed proficient degradation of polIILS as did cells with deficiencies in the recovery of RNA synthesis following UV-irradiation due to defective p53. Furthermore, we found that proteasome function was important for the recovery of mRNA synthesis even in TCR-deficient HCT116 cells. Our results suggest that proteasome-mediated degradation of polIILS is preceded by phosphorylation of the C-terminal domain of polIILS and requires the CS-A and CS-B but not MLH1 or p53 proteins. Furthermore, our results suggest that following UV-irradiation, the degradation of polIILS is required for the efficient recovery of mRNA synthesis but not for TCR per se.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Differential transcriptional regulation of individual TCR V beta segments before gene rearrangement.

The promoter sequences of individual murine TCR Vbeta segments are dissimilar, but any functional differences between them are masked after productive gene rearrangement by the dominance of the TCRbeta 3' enhancer. However, thymocytes of recombination-activating gene-2 (Rag2)-deficient mice allow the transcriptional activity of Vbeta promoters to be studied before rearrangement. Here we report that many Vbeta segments are detectably transcribed in Rag2(-/-) thymocytes and that there are significant differences in expression among different Vbeta segments. Primer extension and characterization of cDNA clones from SCID thymocytes suggest that these germline Vbeta transcripts generally use the same start sites as those previously determined in mature T cells. The strength of expression before rearrangement does not correlate with proximity to the known enhancer, because members of the most distal Vbeta cluster (Vbeta2.1, Vbeta1.1, Vbeta4.1) are relatively strongly expressed and more proximal Vbeta segments (Vbeta14.1, Vbeta3.1, Vbeta7.1, Vbeta6.1) are only weakly expressed. Different Vbeta segments also show different developmental programs of activation in different thymocyte subsets, with the Vbeta5.1(L)-8.2(V) spliced transcript expressed earliest as well as most strongly overall. Comparison with Rag(+) MHC class I(-/-) and class II(-/-) thymocytes confirms that many of these expression differences are leveled by rearrangement and/or by beta selection, before MHC-dependent selection. However, the expression pattern of Vbeta2.1 is highly distinctive and includes cell types apparently outside the T lineage, suggesting potential acquisition of specialized roles.

Animals↗

PubMed2001

Opposite effect of NF-kappa B and c-Jun N-terminal kinase on p53-independent GADD45 induction by arsenite.

Cell cycle checkpoint, a major genomic surveillance mechanism, is an important step in maintaining genomic stability and integrity in response to environmental stresses. Using cells derived from human bronchial epithelial cells, we demonstrate that NF-kappaB and c-Jun N-terminal kinase (JNK) reciprocally regulate arsenic trioxide (arsenite)-induced, p53-independent expression of GADD45 protein, a cell cycle checkpoint protein that arrests cells at the G(2)/M phase transition. Inhibition of NF-kappaB activation by stable expression of a kinase-mutated form of IkappaB kinase caused increased and prolonged induction of GADD45 by arsenite. In contrast, the induction of GADD45 by arsenite was transient and less potent in cells where the NF-kappaB activation pathway was normal. Analysis of the cell cycle profile by flow cytometry indicated that NF-kappaB inhibition potentiates arsenite-induced G(2)/M cell cycle arrest. Abrogation of JNK activation, on the other hand, decreased GADD45 expression induced by arsenite, suggesting a role for JNK activation in GADD45 induction. These results indicate a molecular mechanism by which NF-kappaB and JNK may differentially contribute to cell cycle regulation in response to arsenite.

Arsenites↗

PubMed2001

Preparative isolation and purification of lutein from the microalga chlorella vulgaris by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied to the isolation and purification of lutein from microalgae. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of n-hexane-ethanol-water (4:3:1, v/v). Using the above solvent system, preparative HSCCC was successfully performed yielding lutein at 98% purity from 200 mg of the crude extract in a one-step separation.

Chlorella↗

PubMed2001

Tau filament formation in transgenic mice expressing P301L tau.

Mutations in the microtubule-associated protein tau, including P301L, are genetically coupled to hereditary frontotemporal dementia with parkinsonism linked to chromosome 17. To determine whether P301L is associated with fibril formation in mice, we expressed the longest human tau isoform, human tau40, with this mutation in transgenic mice by using the neuron-specific mouse Thy1.2 promoter. We obtained mice with high expression of human P301L tau in cortical and hippocampal neurons. Accumulated tau was hyperphosphorylated and translocated from axonal to somatodendritic compartments and was accompanied by astrocytosis and neuronal apoptosis indicated by terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling staining. Moreover, P301L tau formed abnormal filaments. Electron microscopy of sarcosyl-insoluble protein extracts established that the filaments had a straight or twisted structure of variable length and were approximately 15 nm wide. Immunoelcecton microscopy showed that the tau filaments were phosphorylated at the TG3, AT100, AT8, and AD199 epitopes in vivo. In cortex, brain stem, and spinal cord, neurofibrillary tangles were also identified by thioflavin-S fluorescent microscopy and Gallyas silver stains. Together, our results show that expression of the P301L mutation in mice causes neuronal lesions that are similar to those seen in human tauopathies.

Actin Cytoskeleton↗