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Biomedical subjects

L Chen

Publications and source records attributed to L Chen.

At least 325 records · Page 18Linked to original sources
PubMed2000

Transferred DNA (T-DNA)-associated proteins of Agrobacterium tumefaciens are exported independently of virB.

The transfer of T-DNA from Agrobacterium to plant cells is mediated by a system which involves the virB operon of the Ti plasmid. We report that VirE2 and VirD2, two T-DNA-associated proteins, as well as VirF, a protein known to be secreted into plant cells, are present in the periplasm and supernatant fractions of growing cells of Agrobacterium as are VirJ and ChvE, two known periplasmic proteins. Two cytoplasmic proteins, Ros and chloramphenicol acetyl transferase, and a VirE2green fluorescent protein construct were not detected in the above fraction. Export of VirE2 into the culture supernatant did not require any Ti plasmid genes, except for VirE1, a specific chaperone for VirE2. The levels of the VirE2 and VirD2 proteins in the supernatant increased significantly when cells were grown at 19 degrees C as compared with 28 degrees C. When Agrobacterium expressed the oncogenic suppressive activity protein (Osa), VirE2 and VirF proteins could not be detected in the supernatant or the periplasm and the level of VirD2 was greatly reduced. However, oncogenic suppressive activity protein did not block the accumulation of VirJ and ChvE in the periplasm. Our data suggest that VirD2, VirE2, and VirF are transported across the cytoplasmic membrane by a specific pathway, independent of virB. Thus, transfer of the T-complex of Agrobacterium may take place in two steps, the first mediated by an unidentified pathway and the second by the virB system.

Agrobacterium tumefaciens↗
PubMed2000

Promoter activity and regulation of the CYP4F2 leukotriene B(4) omega-hydroxylase gene by peroxisomal proliferators and retinoic acid in HepG2 cells.

The human liver CYP4F2 gene (Accession No. AF221943) encodes a leukotriene B(4) omega-hydroxylase that metabolizes leukotriene B(4) (LTB(4)) to a less potent proinflammatory eicosanoid, 20-OH-LTB(4). We sequenced a 6.7-kb genomic fragment of the human CYP4F2 gene that has the first five exons and 500 bp of the 5'-flanking region. The major transcription start site was found to be 49 bp upstream of the 3' end of exon 1 and the ATG translation initiation codon was located in exon 2. Besides the TATA box at -39 bp and basal transcription factor binding sites, the promoter region and 412-bp intron 1 have several putative binding sites for nuclear factors that may mediate the inflammatory response and lipid homeostasis. We found two DR1 elements in the 5' promoter, a DR2 element in intron 1, and RXR/RAR binding sites in both intron 1 and the 5' promoter. DNase I footprinting revealed three protected sequences, with the region containing two CAATT boxes at -71 and -111 bp important in CYP4F2 gene expression. Luciferase reporter assays showed that the 500-bp upstream sequence has strong promoter activity. Transient transfection experiments identified two sites in the 5' promoter and intron 1 that cooperate in gene transcription while exon 1 and a GC-rich region flanking exon 1 inhibit transcription. trans-Retinoic acid and 9-cis-retinoic acid stimulate promoter activity 3- and 6-fold, respectively, while cotransfection with RXRalpha or RAR/RXRalpha further enhanced activity. Peroxisome proliferators inhibit CYP4F2 gene promoter activity and cotransfection with PPARalpha or PPARalpha/RXRalpha can slightly attenuate this inhibition. Both saturated fatty acids and 12-hydroxydodecanoic acid (12-OH-C(12)) can stimulate CYP4F2 gene promoter activity. Therefore, the CYP4F2 gene is repressed by peroxisomal proliferators and induced by retinoic acid, with RAR/RXRalpha mediating the induction while PPARalpha/RXR functions neither in the repression nor in the induction by peroxisomal proliferators or retinoic acid.

Amino Acid Sequence↗
PubMed2000

Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: long- term remission of liver metastases in a mouse model.

BACKGROUND: The success of immunomodulatory cancer therapy is frequently hampered by the transient nature of the antitumor immune response. We have shown previously in a mouse model that interleukin 12 (IL-12) generates a strong natural killer (NK) cell-mediated antitumor response and reduces liver metastases induced by a colon carcinoma cell line. However, only a small percentage of the treated animals developed the cytotoxic T-lymphocytic response required for a long-term systemic antitumor immunity. 4-1BB is a co-stimulatory molecule expressed on the surface of activated T cells. Interaction of 4-1BB with its natural ligand (4-1BBL) has been shown to amplify T-cell (especially CD8+)-mediated immunity. In this study, we investigated the effects of adenovirus-mediated gene therapy delivering both IL-12 and 4-1BBL genes on mice with hepatic metastases induced by colon cancer cells. METHODS: Syngeneic BALB/c mice received intrahepatic injection of poorly immunogenic MCA26 colon cancer cells. Various combinations of replication-defective adenoviruses expressing IL-12 and 4-1BBL genes were injected into the established liver tumors. Changes in tumor size and animal survival were then monitored. All statistical tests were two-sided. RESULTS: The long-term survival rate of mice treated with the combination of IL-12 and 4-1BBL was significantly improved over that of animals in the control group (P =.0001). In vivo depletion of NK cells or CD8+ T cells completely abolished the long-term survival advantage of the IL-12 plus 4-1BBL-treated animals (P<.002). Moreover, the systemic immunity induced by this combination treatment protected these animals against a subcutaneous challenge with parental MCA26 cells. CONCLUSION: Adenovirus-mediated transfer of IL-12 and 4-1BBL genes directly into liver tumors resulted in tumor regression that required both NK and CD8+ T cells and generated a potent, long-lasting antitumor immunity.

4-1BB Ligand↗
PubMed2000

Developmental changes in the expression of NMDA receptor subunits (NR1, NR2A, NR2B) in the cat visual cortex and the effects of dark rearing.

The present study used Western blots to determine changes in the level of expression of the three major NMDA receptor subunits, NR1, NR2A, and NR2B, in relation to the 'critical period' in cat visual cortex. NR2A rose dramatically (10-fold) from very low levels at 1 week to a peak at 5 weeks and gradually declined into adulthood (twofold). NR2B showed a similar time course to NR2A, but the rise from 1 to 5 weeks was of lesser magnitude (twofold). NR1 was expressed at comparable levels at 1, 5, 10 weeks and declined markedly (fivefold) in older animals. No significant effects of dark rearing on the levels of NR2B and NR1 were found. However, NR2A expression was significantly elevated in normal compared to dark reared visual cortex (twofold) at 5 weeks and significantly elevated in dark reared compared to normal visual cortex at 20 weeks (twofold). The close agreement between NR2A expression and both the time course of the critical period and the effects of dark rearing on that time course further indicates a role of this subunit in visual cortical critical period plasticity.

Age Factors↗
PubMed2000

Risk factors for syncope in a community-based sample (the Framingham Heart Study).

The epidemiology of syncope has not been well described. Prior studies have examined risk factors for syncope in hospital-based or other acute or long-term care settings. To determine risk factors for syncope in a community-based sample, we performed a nested case-control study. We examined reports of syncope in Framingham Heart Study participants who underwent routine clinic visits from 1971 to 1990. For each syncope case (n = 543) 2 controls were matched for age, sex, and examination period. Mean age of subjects was 67 years (range 25 to 95); 59% were women. History of stroke or transient ischemic attack, history of myocardial infarction, high blood pressure, use of antihypertensive medication, use of other cardiac medication, smoking, alcohol intake, body mass index, systolic blood pressure, diastolic blood pressure, heart rate, atrial fibrillation, PR interval prolongation, interventricular block, and diabetes or elevated glucose level were examined as potential predictors. Using conditional logistic regression analysis, the predictors of syncope included a history of stroke or transient ischemic attack (odds ratio [OR] 2.56, 95% confidence interval [CI] 1.62 to 4.04), use of cardiac medication (OR 1.67, 95% CI 1.21 to 2. 30), and high blood pressure (OR 1.46, 95% CI 1.14 to 1.88). Lower body mass index was marginally associated with syncope (OR per 4 kg/m(2) decrement 1.10, 95% CI 0.99 to 1.22), as were increased alcohol intake (OR per 5 oz/week 1.11, 95% CI 0.99 to 1.26), and diabetes or an elevated glucose level (OR 1.29, 95% CI 0.96 to 1.75). To our knowledge, this study represents the first community-based study of risk factors for syncope.

Adult↗
PubMed2000

Caenorhabditis elegans beta-G spectrin is dispensable for establishment of epithelial polarity, but essential for muscular and neuronal function.

The Caenorhabditis elegans genome encodes one alpha spectrin subunit, a beta spectrin subunit (beta-G), and a beta-H spectrin subunit. Our experiments show that the phenotype resulting from the loss of the C. elegans alpha spectrin is reproduced by tandem depletion of both beta-G and beta-H spectrins. We propose that alpha spectrin combines with the beta-G and beta-H subunits to form alpha/beta-G and alpha/beta-H heteromers that perform the entire repertoire of spectrin function in the nematode. The expression patterns of nematode beta-G spectrin and vertebrate beta spectrins exhibit three striking parallels including: (1) beta spectrins are associated with the sites of cell-cell contact in epithelial tissues; (2) the highest levels of beta-G spectrin occur in the nervous system; and (3) beta spectrin-G in striated muscle is associated with points of attachment of the myofilament apparatus to adjacent cells. Nematode beta-G spectrin associates with plasma membranes at sites of cell-cell contact, beginning at the two-cell stage, and with a dramatic increase in intensity after gastrulation when most cell proliferation has been completed. Strikingly, depletion of nematode beta-G spectrin by RNA-mediated interference to undetectable levels does not affect the establishment of structural and functional polarity in epidermis and intestine. Contrary to recent speculation, beta-G spectrin is not associated with internal membranes and depletion of beta-G spectrin was not associated with any detectable defects in secretion. Instead beta-G spectrin-deficient nematodes arrest as early larvae with progressive defects in the musculature and nervous system. Therefore, C. elegans beta-G spectrin is required for normal muscle and neuron function, but is dispensable for embryonic elongation and establishment of early epithelial polarity. We hypothesize that heteromeric spectrin evolved in metazoans in response to the needs of cells in the context of mechanically integrated tissues that can withstand the rigors imposed by an active organism.

Amino Acid Sequence↗
PubMed2000

Identification of the human pituitary tumor transforming gene (hPTTG) family: molecular structure, expression, and chromosomal localization.

In an attempt to determine the mechanism of human tumorigenesis, we have searched for oncogenes and recently reported the molecular cloning of a potent oncogene (hPTTG) from human testis. hPTTG mRNA is expressed at high levels in various human tumors and tumor cell lines. Overexpression of hPTTG in the mouse fibroblast cell line (NIH 3T3) results in an increase in cell proliferation, induces cellular transformation in vitro, and promotes tumor formation in nude mice. The hPTTG gene isolated from the human genomic library consists of five exons and four introns and spans over 10kb. In the studies reported here, we further investigated the possibility of the presence of additional genes homologous to hPTTG in the human genome, which was first indicated by Southern blot analysis of the human genomic DNA and chromosomal mapping of the hPTTG gene using DNA from humanxhamster hybrid cell lines in PCR. Sequencing and restriction map analysis of the additional genomic clones identified two intronless genes homologous to hPTTG. This finding was confirmed by the chromosomal location of the second gene to chromosome 4p15.1 and the third gene to chromosome 8q13.1. Based on the similarity in sequences, we proposed that hPTTG be renamed hPTTG1 and the new genes be named hPTTG2 and hPTTG3. hPPTG2 was found to be 91% identical and hPPTG3 89% identical with hPPTG1 at the amino acid level. Northern blot and reverse transcriptase/polymerase chain reaction (RT/PCR) analyses of the mRNA from various human tissues revealed differential expression of the hPTTG2 and hPTTG3 genes in normal and tumor tissues, suggesting that these genes may be associated with tumorigenesis.

Amino Acid Sequence↗
PubMed2000

N-benzylpyroglutamyl-L-phenylalanine derivatives as VCAM/VLA-4 antagonists.

A series of N-(N-benzylpyroglutamyl)-4-substituted-L-phenylalanine derivatives was prepared as VLA-4/VCAM antagonists. Analogues substituted by electron deficient benzoylamino groups bearing bulky ortho substituents had low-nM potency in an ELISA assay and low-microM activity in a cell based assay.

Cell Line↗
PubMed2000

Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue improves the 3'-exonuclease stability of 2'-5'-oligoadenylate-antisense conjugates.

Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue at the 3'-terminus of DNA or 2-5A-DNA sequences resulted in a significantly enhanced 3'-exonuclease resistance while the affinity for complementary RNA was only slightly decreased. Furthermore, the binding to and activation of human RNase L by thus modified 2-5A-DNA conjugates was not altered as compared to the parent unmodified 2-5A-DNAs.

Adenine Nucleotides↗
PubMed2000

LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response.

LIGHT is a recently identified member of the TNF superfamily and its receptors, herpesvirus entry mediator and lymphotoxin beta receptor, are found in T cells and stromal cells. In this study, we demonstrate that LIGHT is selectively expressed on immature dendritic cells (DCs) generated from human PBMCs. In contrast, LIGHT is not detectable in DCs either freshly isolated from PBMCs or rendered mature in vitro by LPS treatment. Blockade of LIGHT by its soluble receptors, lymphotoxin beta receptor-Ig or HVEM-Ig, inhibits the induction of DC-mediated primary allogeneic T cell response. Furthermore, engagement of LIGHT costimulates human T cell proliferation, amplifies the NF-kappaB signaling pathway, and preferentially induces the production of IFN-gamma, but not IL-4, in the presence of an antigenic signal. Our results suggest that LIGHT is a costimulatory molecule involved in DC-mediated cellular immune responses.

Amino Acid Sequence↗
PubMed2000

Overexpression, purification, and analysis of complementation behavior of E. coli SuhB protein: comparison with bacterial and archaeal inositol monophosphatases.

The E. coli suhB gene product, which has been suggested to participate in posttranscriptional control of gene expression, also possesses inositol-1-phosphatase (I-1-Pase) activity. To test if SuhB I-1-Pase activity is sufficient for its function in cells, we have cloned the genes for three other I-1-Pases (from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus, and from the bacterium Thermotoga maritima) into the E. coli expression vector pET23a(+) and examined if these extragenic I-1-Pases could complement the suhB mutation in E. coli strain CG1307 (which also has a mutation in dnaB and a cold-sensitive phenotype). None of these I-1-Pase genes restored growth at 30 degrees C although they generated active I-1-Pase enzymes (as measured by I-1-Pase specific activities of crude protein extracts from the transformed CG1307 cells). In contrast, the pET23a(+) recombinant plasmid with the wild-type E. coli suhB gene complemented the cold sensitivity of the chromosomal mutant suhB and restored the temperature-sensitive growth of the dnaB mutation in the double mutant strain CG1307. Further evidence that this relief of the suppressor behavior of the suhB mutation is not related to the I-1-Pase activity of the SuhB protein was provided by construction of the E. coli SuhB mutant D87N. This mutant protein is inactive as an I-1-Pase but fully functional in changing the temperature sensitivity of the E. coli double mutant strain. Therefore, I-1-P phosphatase activity is neither sufficient nor required for complementation of suhB mutant suppressor effects. The wild-type E. coli SuhB protein was also overexpressed to very high levels and purified to homogeneity in high yield (1 mg/10 mL of culture). The major differences of the E. coli I-1-Pase from all the other characterized I-1-Pases are that it exists as a monomer (rather than a dimer or tetramer) in solution and is more hydrophobic. These physical differences, rather than the I-1-Pase activity, may be involved in the biological role of wild-type SuhB in E. coli.

Amino Acid Sequence↗
PubMed2000

Haploid loss of the tumor suppressor Smad4/Dpc4 initiates gastric polyposis and cancer in mice.

The tumor suppressor SMAD4, also known as DPC4, deleted in pancreatic cancer, is a central mediator of TGF-beta signaling. It was previously shown that mice homozygous for a null mutation of Smad4 (Smad4-/-) died prior to gastrulation displaying impaired extraembryonic membrane formation and endoderm differentiation. Here we show that Smad4+/- mice began to develop polyposis in the fundus and antrum when they were over 6 - 12 months old, and in the duodenum and cecum in older animals at a lower frequency. With increasing age, polyps in the antrum show sequential changes from hyperplasia, to dysplasia, in-situ carcinoma, and finally invasion. These alterations are initiated by a dramatic expansion of the gastric epithelium where Smad4 is expressed. However, loss of the remaining Smad4 wild-type allele was detected only in later stages of tumor progression, suggesting that haploinsufficiency of Smad4 is sufficient for tumor initiation. Our data also showed that overexpression of TGF-beta1 and Cyclin D1 was associated with increased proliferation of gastric polyps and tumors. These studies demonstrate that Smad4 functions as a tumor suppressor in the gastrointestinal tract and also provide a valuable model for screening factors that promote or prevent gastric tumorigenesis.

Age Factors↗
PubMed2000

The role of ClC-3 in volume-activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition.

1. A chloride current with mild outward rectification was induced in the native bovine non-pigmented ciliary epithelial (NPCE) cells by a 23 % hypotonic solution. The current showed no or little inactivation at depolarized steps. 2. ATP blocked 88 and 61 % of the outward and inward components of the volume-activated chloride current (ICl,vol) with an IC50 of 5.3 and 9.6 mM, respectively. 3. The volume-activated chloride current was decreased and the activation of the current was delayed by inhibiting endogenous ClC-3 expression using a ClC-3 antisense oligonucleotide. The inhibition of the current as a function of antisense concentration was asymptotic with a maximum about 60 %. The remaining current was probably not derived from ClC-3 and was inhibited by ATP. 4. ClC-3 expression in the bovine NPCE cells was verified by immunofluorescence studies. ClC-3 immunofluorescence was distributed throughout the cells but with the predominant location within the nucleus. The expression of ClC-3 protein was diminished by the ClC-3 antisense oligonucleotide with the greatest diminution occurring in the nuclear region. 5. The size of the volume-activated chloride current was positively correlated with the ClC-3 immunofluorescence level. 6. Regulatory volume decrease of the NPCE cells was reduced by ClC-3 antisense oligonucleotide. 7. We conclude that endogenous ClC-3 is associated with the volume-activated chloride current and is involved in cell volume regulation, but that it can only contribute towards a proportion of the current in NPCE cells. 8. The nuclear predominance of ClC-3 immunofluorescence in NPCE cells, the absence of basal activity of chloride current and the marked pharmacological differences between IClC-3 and ICl,vol argue against ClC-3 being the only, or even the main, volume-activated chloride channel in NPCE cells.

Adenosine Triphosphate↗
PubMed2000

In vitro growth inhibition of a broad spectrum of tumor cell lines by activated human dendritic cells.

Dendritic cells (DCs) are critical subsets of leukocytes providing antigen presentation for initiation of humoral and cellular immune responses. Their role as effector cells in tumor resistance, however, is less known. We report here that human DCs generated by culturing plastic-adherent peripheral blood monocytes in the presence of granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin-4 have potent growth-inhibition activity in vitro on a wide spectrum of human tumor lines of different tissue origin. Proinflammatory stimuli lipopolysaccharide (LPS) and interferon-gamma, but not tumor necrosis factor-alpha and CD40 signaling, can further enhance DC-mediated inhibition of tumor growth. The growth inhibition requires contact between DCs and tumor cells while LPS treatment enhances the antitumor activity in DC culture supernatants. Our results suggest that in addition to their predominant role as regulatory cells, activated DCs are also potential effector cells in tumor immunity.

Cell Adhesion↗
PubMed2000

Antitumor activity of endostatin against carcinogen-induced rat primary mammary tumors.

Endostatin, a Mr 20,000 fragment of collagen XVIII, potently inhibits the growth of experimental tumors implanted in mice. Here we report the cloning, expression, and antitumor activity of the rat form of endostatin. When tested on breast carcinomas arising in female virgin rats after intragastric administration of 9,10-dimethyl-1,2-benzanthracene (DMBA), endostatin induced significant inhibition of mammary tumor growth in all of the treated rats during a 4-week treatment period without signs of systemic toxicity. Interestingly, this arrest of tumor growth persisted throughout a four-week off-therapy period. Moreover, endostatin was effective in counteracting the development of multiple primary tumors. These results confirm that rat endostatin is a potent anticancer agent in a carcinogen-induced, spontaneously arising rat breast cancer model. It not only stops the growth of existing tumors but also decreases the incidence of the development of multiple neoplastic lesions.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2000

Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803.

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro trans-splicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the N- and C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.

Base Sequence↗