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Biomedical subjects

A Chen

Publications and source records attributed to A Chen.

At least 163 records · Page 9Linked to original sources
PubMed2000

[Zafirlukast inhibition of leukotriene C4 induced endothelin-1 expression in human airway structural cell].

OBJECTIVE: To investigate the effects of a cysteinyl leukotriene receptor antagonist (zafirlukast) on endothelin-1(ET-1) expression in human airway epithelial cell line (16-HBE) and airway smooth muscle cells (ASMC) in response to leukotriene C4(LTC4) stimulation. METHODS: 16-HBE or ASMC was incubated with 10(-8) mol/L LTC4 and graded concentrations of zafirlukast. ET-1 level in supernatant was determined by ELISA and ET-1 mRNA expression was assessed by RT-PCR. RESULTS: (1) LTC4 induced ET-1 mRNA expression in 16-HBE and ASMC was significantly inhibited by zafirlukast (10(-8) mol/L) 0.23 +/- 0.10 vs. 0.10 +/- 0.03 (16-HBE), t = 3.698, P = 0.034; 0.13 +/- 0.04 vs. 0.04 +/- 0.02 ASMC, t = 4.629, P = 0.019. (2) When graded concentrations of zafirlukast (0, 10(-12) mol/L, 10(-10) mol/L, 10(-8) mol/L, 10(-6) mol/L) were added to the LTC4 cultured 16-HBE and ASMC, a significant inhibition of ET-1 production was observed in both groups cultured with 10(-8) mol/L or more zafirlukast [(12.7 +/- 1.3) ng/L vs (5.5 +/- 2.6) ng/L 16-HBE, t = 4.693, P = 0.019; (7.5 +/- 1.0) ng/L vs (4.4 +/- 0.9) ng/L ASMC (t = 5.003, P = 0.007)]. (3) There was a significant negative linear correlation between ET-1 levels and zafirlukast concentrations in LTC4-treated 16-HBE and ASMC (16-HBE: r = -0.9177, P = 0.0289; ASMC: r = -0.9451, P = 0.0153). CONCLUSIONS: These data suggest that zafirlukast inhibit LTC4-induced ET-1 over-expression 16-HBE and ASMC, which may be one of the anti-inflammatory mechanism of LTs receptor antagonists.

Endothelin-1↗
PubMed2000

[Expression and characterization of human vascular endothelial growth factor in Pichia pastoris].

OBJECTIVE: To study the expression of human VEGF165 cDNA in Pichia pastoris and to obtain high-level expression of recombinant human VEGP165 (hVEGF165) with good biological activity. METHODS: Amplifying hVEGF165 cDNA by PCR, after confirmed by DNA sequence analysis, the gene was inserted into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and a secreting signal peptides, the recombinant expression plasmid pPIC9K/VEGF165 was constructed and transformed into KM71. The multiple insert transformants were screened, fermented in flasks and induced by 1% methanol. RESULTS: After 4 days of methanol induction, the expressed hVEGF165 came up to 30% of total proteins in supernatant by SDS-PAGE. The expressed hVEGF165 was further proved having good antigenicity and high specificity by ELISA and Western blot assay, and having good biological activity to stimulate HUVEC proliferation. CONCLUSIONS: High-level expression of secreted hVEGF165 had been successfully achieved in Pichia pastoris expression system.

Endothelial Growth Factors↗
PubMed2000

[Clinical study on chitosan for prevention of elbow adhesion].

OBJECTIVE: To study the clinical effect of chitosan on prevention of elbow adhesion after elbow arthrolysis. METHODS: Twenty six patients with elbow ankylosis were performed elbow arthrolysis, which divided into two groups, in chitosan group, 12 patients were injected 2% chitosan into the elbow joint cavity, and no chitosan used in the other 14 patients as control group. The average range of extension and flexion of elbow joint was detected to evaluate the clinical results. RESULTS: All patients were followed up 8 to 51 months, averaged 24 months. In the chitosan group, the average range of extension and flexion of elbow joint was restored to 92.9 degrees +/- 20.9 degrees, with an average increase of 55.0 degrees +/- 15.9 degrees compared with preoperation. In the control group, the average range of extension and flexion of elbow joint was restored to 75.4 degrees +/- 17.5 degrees, with an average increase of 38.2 degrees +/- 11.9 degrees. The outcome showed significant difference between the chitosan group and the control group (P < 0.01). CONCLUSION: Chitosan can prevent or reduce elbow adhesion after elbow arthrolysis.

Adolescent↗
PubMed2000

[Experimental and clinical study of gentamicin-loaded chitosan drug delivery system].

OBJECTIVE: To conduct a clinical study and release test for a drug delivery system (DDS) of gentamicin-loaded chitosan bar in attempt to extend its clinical application. METHODS: The diffusion behavior of gentamicin from chitosan DDS was detected using in vitro, in vivo release test. Eighteen cases of chronic osteomyelitis were treated by chitosan DDS. The clinical result was evaluated by wound healing and clinical and X-radiographic manifestation. RESULTS: Concentration of gentamicin about 926.7 microg/bar/day was released from DDS within the first 24 hours by in vitro diffusion test. It fell and sustained by 25th day with rate of 4.0 microg x bar(-1) x day(-1). In in vivo study, serum gentamicin concentration reached its peak level (0.92 microg/ml) 24 hours after implantation. No increase was observed in the concentration of BUN and Cr. In all bone tissue around the bar, 8 weeks after oderation the gentamicin concentration exceeded the minimum inhibitory concentration for the common causative organisms of osteomyelitis. The follow up time was 24.8 months (6 - 34). The initial cure ratio was 2/18 and the recurrence ratio zero. CONCLUSION: The gentamicin-loaded chitosan bar is a good DDS with sustained antibiotic effect in vivo and in vitro. It is an effective method for the treatment of bone infection.

Animals↗
PubMed1999

The gonadotropin-releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform.

The mammalian gonadotropin-releasing hormone (GnRH-I), which regulates reproduction, was the first isoform of GnRH that was identified in mammals. Recently, we and others have demonstrated the existence of a second isoform of GnRH in the brain of mammals. The presence of a third isoform of GnRH, GnRH-III, in the brain of mammals is reported herein. GnRH-III, extracted from the brain of bovine and human, was purified by high performance liquid chromatography, using two distinct elution programs. In both, GnRH-III was eluted at the same positions as synthetic salmon GnRH, as demonstrated by radioimmunoassay. The luteinizing hormone-releasing activity of purified GnRH-III, using dispersed rat pituitary cells, was found to be similar to that of synthetic salmon GnRH. The total amount of GnRH-III, determined by radioimmunoassay, in the hypothalamus and midbrain of humans and calves is similar to that of GnRH-I. Immunohistochemical studies demonstrated GnRH-III-containing neurons in the hypothalamus and midbrain of human and GnRH-III fibers in the median eminence of rats. The distribution of GnRH-III in the brain suggests that in addition to a putative function as a neurohormone at the hypothalamic-pituitary axis, GnRH-III may have other functions. Our present results suggest that multiple isoforms of GnRH are present in the brain of mammals, and further studies are required in order to elucidate their biological functions.

Amino Acid Sequence↗
PubMed1999

1,25-Dihydroxyvitamin D(3) stimulates activator protein-1-dependent Caco-2 cell differentiation.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a potential chemopreventive agent for human colon cancer. We have reported that 1,25(OH)(2)D(3) specifically activated protein kinase C-alpha (PKC-alpha) and also caused a reduction in proliferation while increasing apoptosis and differentiation in CaCo-2 cells, a cell line derived from a human colon cancer. The mechanisms by which this secosteroid influences these important cellular processes, however, remain unclear. The transcription factor, activator protein-1 (AP-1), regulates many genes involved in these processes. Therefore, we asked whether 1,25(OH)(2)D(3) activated AP-1 in CaCo-2 cells and, if so, by what mechanisms? 1,25(OH)(2)D(3) caused a time-dependent increase in AP-1 DNA binding activity and significantly enhanced the protein and mRNA abundance of c-Jun, a component of AP-1. 1, 25(OH)(2)D(3) also induced a rapid and transient activation of ERK2 (where ERK is extracellular signal-regulated kinase) and a more persistent activation of JNK1 (where JNK Jun N-terminal kinase). Transfection experiments revealed that 1,25(OH)(2)D(3) also increased AP-1 gene-transactivating activity. This AP-1 activation was completely blocked by PD 098059, a specific mitogen-activated protein kinase/ERK kinase inhibitor, as well as by a dominant negative JNK or a dominant negative Jun, indicating that the AP-1 activation induced by 1,25(OH)(2)D(3) was mediated by ERK and JNK. Using a specific inhibitor of the Ca(2+)-dependent PKC isoforms, Gö6976, and CaCo-2 cells stably transfected with antisense PKC-alpha cDNA, demonstrated that PKC-alpha mediated the AP-1 activation induced by this secosteroid. Inhibition of JNK activation or c-Jun protein expression significantly reduced 1, 25(OH)(2)D(3)-induced alkaline phosphatase activity, a marker of CaCo-2 cell differentiation, in secosteroid-treated cells. Taken together, the present study demonstrated that 1,25(OH)(2)D(3) stimulated AP-1 activation in CaCo-2 cells by a PKC-alpha- and JNK-dependent mechanism leading to increases in cellular differentiation.

Caco-2 Cells↗
PubMed1999

Affinity NMR.

Because binding ligands are directly detected and identified, the diffusion-based NMR method and NOE pumping approach promise to greatly simplify deconvolution in drug screening. An additional advantage of these techniques is that low-affinity ligands, which might be missed by high-throughput screening, can be detected and could serve as synthetic precursors for higher affinity ligands. The biggest challenge to NMR methodology lies in its sensitivity. Compared with other techniques, such as MS (25), NMR methods for screening mixtures are limited by their relative insensitivity. Because of issues such as solubility, stability, and mass limitation, it is not in general judicious to simply increase the concentration of the mixture. Improvements in hardware and software are necessary to extend the applicability of the affinity NMR method to the screening of larger and more complex mixtures. A boost in sensitivity and screening capacity of NMR technique is possible by the implementation of microcoil (26) and flow probe techniques. An upsurge in the capabilities of mixture analysis could be achieved with a combination of independent and complementary techniques (e.g., HPLC, MS) (27). As a unique, nondestructive, and versatile tool, NMR will continue on its fast track of development in the support of drug discovery.

Drug Evaluation, Preclinical↗
PubMed1999

Intraoperative 125I brachytherapy for high-risk stage I non-small cell lung carcinoma.

PURPOSE: Preliminary assessment of feasibility, efficacy, acute and chronic side effects associated with permanent intraoperative placement of 125I vicryl mesh brachytherapy in a select group of high-risk Stage I NSCLC who have undergone video-assisted thoracoscopic resection (VATR). METHODS AND MATERIALS: From January 8, 1997 to March 16, 1998, 23 patients with Stage I NSCLC at high risk for conventional surgery due to cardiopulmonary compromise underwent combined VATR and intraoperative placement of 125I seeds embedded in vicryl mesh. Seeds embedded in vicryl suture were attached with surgical clips to a sheet of vicryl mesh, and thoracoscopically inserted over the target area (tumor bed and staple line) with nonabsorbable suture or surgical clips. A total dose of 100-120 Gy prescribed to the periphery of the target area (defined as the staple line and tumor bed with a 1-cm margin) was delivered. RESULTS: The mean target area covered was 48 cm2 (range 40-72) and mean total activity was 22 mCi (range 17.2-28.2). The median length of postoperative stay was 7 days. The median follow-up was 11 months (range 2-20). Postoperative CT scans of the chest revealed no dislodgement of the seeds and no local recurrence in any patient. Three patients developed distant metastasis (1 died 6 months postoperatively; the other 2 are currently alive with disease). One patient developed an ipsilateral recurrence in the right lower lobe after having had a right upper lobe resection. There were 3 postoperative deaths due to medical comorbid conditions or surgical complications (1 in the immediate postoperative period). Pulmonary function testing performed 3 months after implantation revealed no significant difference between preoperative and postoperative values: mean preoperative FVC was 2.3 L (range 1.31-3.0) and postoperative FVC was 2.2 L (range 1.1-3.9), p = 0.42; mean preoperative FEV1 was 1.2 L (range 0.71-2.2), and postoperative FEV1 was 1.5 L (range 0.8-2.9), p = 0.28. CONCLUSION: Review of early data suggests that intraoperative 125I vicryl mesh brachytherapy in high-risk Stage I NSCLC is potentially effective and well tolerated, with no significant decline in measurable pulmonary function studies and no increase in postoperative complications. Longer follow-up is needed to determine ultimate local control and survival.

Aged↗
PubMed1999

Reduction in cell size during development of the spinal cord.

During development, spinal cord cells of the frog Xenopus laeuis undergo a reduction in size. This phenomenon occurs during neural tube formation and continues at least until the start of metamorphosis. The number and shape of spinal cord cells also changes, but not always in synchrony with the reduction in cell size. The concomitant change in size and number of spinal cord cells during embryogenesis suggests that a cleavage type of reductive division contributes to the decrease in cell size. Blocking cell division with a combination of hydroxyurea and aphidicolin (HUA) stops the decrease in cell size during embryonic development without affecting the differentiation of a specific class of catecholaminergic neurons. HUA treatment during larval stages does not block the decrease in catecholaminergic neuron size. Thus, both mitotic and postmitotic cells decrease in size during spinal cord development. The two mechanisms are prevalent at different developmental stages with reductive division and cellular atrophy common during embryonic and larval phases, respectively. Like other regressive changes such as cell death and synapse elimination, decreases in cell size affect spinal cord morphogenesis and presumably the function of developing spinal cord cells.

Animals↗
PubMed1999

NIMA-related kinases: isolation and characterization of murine nek3 and nek4 cDNAs, and chromosomal localization of nek1, nek2 and nek3.

The Aspergillus NIMA kinase plays a key role in controlling entrance into mitosis, and recent evidence suggests that mammalian NIMA-related kinases perform similar functions. We report here the cloning of the mouse nek3 and nek4 genes. Mouse nek3 is probably the ortholog of the partially sequenced, human nek3, whereas murine nek4 cDNA is probably the ortholog of human STK2. Nek4 is highly conserved between mouse and human, whereas Nek3 is somewhat less conserved (96.5 and 88% identity in the kinase domains, respectively). Northern analysis shows preferential expression of nek3 in mitotically active tissue, whereas nek4 is highly abundant in the testis. Within the developing testicular germ cells, in-situ analysis demonstrated that nek1, 2 and 4 exhibit differential patterns of expression, suggesting overlapping, but non-identical functions. Linkage analysis, using the mouse recombinant inbred strain panel (BXD), was used to localize nek1, 2 and 3. nek1 was mapped between Cpe and D8Mit8 on chromosome 8 at around 32cM, nek2 was mapped to the distal region of chromosome 1, and nek3 was mapped to the most centromeric region of chromosome 8.

Amino Acid Sequence↗
PubMed1999

CD28 costimulation augments IL-2 secretion of activated lamina propria T cells by increasing mRNA stability without enhancing IL-2 gene transactivation.

The pathways leading to activation in lamina propria (LP) T cells are different from peripheral T cells. LP T cells exhibit enhanced IL-2 secretion when activated through the CD2 pathway. Coligation of CD28 leads to synergistic enhancement of IL-2 secretion. Previous studies have characterized the CD28 augmentation of TCR-mediated signaling in peripheral blood T cells through transcriptional activation of an IL-2 promoter CD28 response element (CD28RE), along with enhanced mRNA stability. This study characterized molecular events involved in CD28 costimulation of IL-2 production in LP mononuclear cells (LPMC). LPMC exhibited increased IL-2 production in response to CD28 costimulation, compared with cells activated through CD2 alone. IL-2 secretion was paralleled by increased expression of IL-2 mRNA, resulting from enhanced IL-2 mRNA stability. In contrast to transcriptional activation in PBMC, EMSA revealed that CD28 coligation of CD2-activated LPMC does not result in increased binding of trans-factors to the CD28RE, nor did Western blots detect changes in I-kappaBalpha or I-kappaBbeta levels following CD28 coligation. Furthermore, CD28 coligation fails to enhance IL-2 promoter-reporter or RE/AP construct expression in CD2-activated LPMC. The results reported herein indicate that the molecular mechanisms involved in CD28 cosignaling and regulation of IL-2 secretion in LP T cells are unique to that compartment and differ from those seen in peripheral blood T cells. These observations suggest a biological significance for different mechanisms of IL-2 activation in initiation and maintenance of the cytokine repertoire found in the mucosa.

Adjuvants, Immunologic↗
PubMed1999

Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression.

Microsatellite instability (MSI) has been described in tumors from patients with hereditary nonpolyposis colorectal cancer, sporadic colorectal cancer, and other types of cancers. MSI is caused by the dysfunction of mismatch repairs genes. Loss of expression and mutation in one of the major mismatch repair genes, hMLH1, and the methylation of CpG sites in its promoter occur frequently in primary tumors and cell lines of colorectal cancer with MSI. To understand the mechanisms involved in the silencing of hMLH1 expression by methylation, we examined the methylation status of all CpG sites in the hMLH1 promoter in 24 colorectal cancer cell lines by the NaHSO3-sequencing method. We identified a small proximal region (-248 to -178, relative to the transcription start site) in the promoter in which the methylation status invariably correlates with the lack of hMLH1 expression. This correlation was further supported by the observation that cell lines that showed methylation-suppressed hMLH1 expression can be induced to reexpress hMLH1 by a methyl transferase inhibitor, 5-aza-2'-deoxycytidine, and the small region that we identified exhibited significant demethylation in all cell lines examined.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Nuclear magnetic resonance chromatography: applications of pulse field gradient diffusion NMR to mixture analysis and ligand-receptor interactions.

Pulse field gradient (PFG) diffusion NMR spectroscopy is a non-invasive method for the spectroscopic separation and identification of compounds of interest from a mixture. Because it relies on differences in translational diffusion rates to resolve NMR signals from individual components, pulse field gradient NMR is a unique method for analyzing complex mixtures and for detecting intermolecular interactions. A number of multidimensional pulse field gradient NMR experiments have been developed to alleviate the overlap of NMR signals arising from a complex mixture and facilitate component identification. The applications of pulse field gradient NMR for mixture analysis and for the direct identification of high affinity ligands are reviewed.

Base Sequence↗
PubMed1999

HOS, a human homolog of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of IkappaB and beta-catenin.

SCF E3 ubiquitin ligases mediate ubiquitination and proteasome-dependent degradation of phosphorylated substrates. We identified a human F-box/WD40 repeats protein (HOS), which is homologous to Slimb/h betaTrCP. Being a part of SCF complex with Skp1 and Cullin1, HOS specifically interacted with the phosphorylated IkappaB and beta-catenin, targeting these proteins for proteasome-dependent degradation in vivo. This targeting required Cullin1 as expression of a mutant Cullin1 abrogated the degradation of IkappaB and of beta-catenin. Mutant HOS which lacks the F-box blocked TNF alpha-induced degradation of IkappaB as well as GSK3beta-mediated degradation of beta-catenin. This mutant also inhibited NF-kappaB transactivation and increased the beta-catenin-dependent transcription activity of Tcf. These results demonstrate that SCF(HOS) E3 ubiquitin ligase regulate both NF-kappaB and beta-catenin signaling pathways.

Amino Acid Sequence↗
PubMed1999

Genomic sequence and structural organization of mouse slow skeletal muscle troponin T gene.

Three muscle type-specific troponin T (TnT) genes are present in vertebrate to encode a number of protein isoforms via alternative mRNA splicing. While the genomic structures of cardiac and fast skeletal muscle TnT genes have been documented, this study cloned and characterized the slow skeletal muscle TnT (sTnT) gene. Complete nucleotide sequence and genomic organization revealed that the mouse sTnT gene spans 11.1kb and contains 14 exons, which is smaller and simpler than the fast skeletal muscle and cardiac TnT genes. Potentially representing a prototype of the TnT gene family, the 5'-region of the sTnT gene contains fewer unsplit large exons, among which two alternatively spliced exons are responsible for the NH2-terminal variation of three sTnT isoforms. The sTnT gene structure shows that the alternatively spliced central segment found in human sTnT cDNAs may be a result from splicing using an alternative acceptor site at the intron 11-exon 12 boundary. Together with the well-conserved protein structure, the highly specific expression of sTnT in slow skeletal muscles indicates a differentiated function of this member of the TnT gene family. The determination of genomic structure and alternative splicing pathways of sTnT gene lays a foundation to further understand the TnT structure-function evolution as well as contractile characteristics of different types of muscle fiber.

Alternative Splicing↗
PubMed1999

Identification of differentially expressed mRNAs in human fetal liver across gestation.

Differential gene expression, with its precise start and stop times, is believed to be critical for the programmed development of new cells and tissues. Within the developing fetus, one tissue of particular interest is fetal liver. This organ undergoes rapid changes in the pathway toward liver development in utero since it is also the major site of hematopoiesis, until bone marrow hematopoiesis predominates. Believing that patterns would emerge from the bi-weekly large-scale inspection of expressed genes in the fetal liver, we employed differential display reverse transcription-polymerase chain reaction (DDRT-PCR) as ourprimary inspection tool. Using DDRT-PCR, we isolated cDNAs differentially expressed throughout fetal liver development and in adult liver. We displayed approximately 25 000 cDNAs from 10 and 24 week fetal liver and adult liver. From this initial screen, we determined that approximately 0.1-1% of the mRNA population undergoes expression changes. We extracted, purified and sequenced 25 differentially displayed cDNA bands. Fourteen cDNAs had similarities to known genes, while 11 cDNAs were not similar to any characterized gene. The differentially expressed cDNAs from known genes present in fetal liver include alpha-fetoprotein, stem cell factor, erythroid alpha-spectrin, 2,3-bisphosphoglycerate mutase, insulin-like growth factor-2, porphobilinogen deaminase and Mac30. The differentially expressed cDNAs present in adult liver but not in 10 week fetal liver were nicotinamide deaminase, human fibrinogen-related protein and alpha-acid glycoprotein. The majority of differentially expressed genes found during this effort appear to be turned on during organogenesis, however, some genes were found that are apparently turned off completely.

Adult↗
PubMed1999

Nerve growth factor and proprotein convertases furin and PC7 in transected sciatic nerves and in nerve segments cultured in conditioned media: their presence in Schwann cells, macrophages, and smooth muscle cells.

Synthesis of proteins such as nerve growth factor (NGF) is induced after nerve lesion. The NGF precursor (pro-NGF) requires a posttranslational processing by proprotein convertases to become active. In this report, we re-examine the localization of NGF protein and mRNA in injured nerve and show that the candidate pro-NGF convertases furin and PC 7 colocalize with NGF in non-neuronal cells in nerve. By Northern blot analysis, 1.5-kb and 1.3-kb NGF mRNAs were shown to be increased in distal and immediately proximal nerve segments on days 1, 4, and 14 after lesion; by Western blot analysis, NGF proteins of high molecular weight were detected after injury. In vivo, two phases of NGF immunopositivity were observed, in macrophages and perivascular cells shortly after lesion and in endoneurial cells on day 1 and 4. To identify the cells containing NGF, nerve segments were incubated in serum-containing medium with or without conditioning by white blood cells isolated from the circulation. Both hybridization and immunoreactivity signals for NGF were elevated after incubation of nerve segments for 4 hours in conditioned media, so that cells with NGF immunoreactivity could be identified by antibodies to specific cell markers. In these nerve fragments, Schwann cells, perivascular smooth muscle cells, and macrophages contained NGF immunoreactivity. The concentration of furin and PC7 mRNA also increased in lesioned nerves. By immunocytochemical investigation of nerve explants, furin and PC7 were detected in endoneurial cells, macrophages and perivascular cells and were colocalized with NGF. These in vitro and in vivo findings suggest that both furin and PC7 are associated with NGF in several cell types of the sciatic nerve and, hence, may be implicated in intracellular processing of pro-NGF.

Animals↗