Biomedical subjects

X Cao

Publications and source records attributed to X Cao.

At least 145 records · Page 8Linked to original sources

PubMed2000

[Research developments of Raman scattering of carbon nanotubes].

Carbon nanotubes is a new one-dimension carbon material. With novel structural, electronic and mechanical properties, carbon nanotubes constitute' an important form of carbon that may find applications in many fields. Raman scattering is an important measurement method for the carbon nanotubes, especially for single-walled carbon nanotubes (SWNTs). The tube configuration can be studied in detail using Raman spectroscopy. Theoretical calculations predicated that SWNTs have 15 or 16 Raman active vibrational modes; the exact number depends on the symmetry of the tubes but is independent of its diameter. Also, the wavenumber of radius breathing mode (RMB) is inverse proportional to the radius of SWNTs by means of Raman spectroscopy. The recent progress of Raman scattering of carbon nanotubes is reviewed in this paper. The characteristic and vibrational theory of carbon nanotubes are presented. The potential application of carbon nanotubes is also briefly described.

Nanotubes, Carbon↗

PubMed1999

Model-based small area estimates of overweight prevalence using sample selection adjustment.

Using a hierarchical model with an adjustment for sample selection, we estimate the overweight prevalence for adults, by states, using data from the Third National Health and Nutrition Examination Survey (NHANES III). A two-stage hierarchical model was selected to account for geographic variability of outcomes and to model possible overdispersion of estimates due to cluster sampling. We compare our model-based estimates with design-based estimates at the national level and obtain excellent agreement. We also provide a check of our model at the state level by comparing estimates with design-based and synthetic estimates.

Adult↗

PubMed1999

Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p.

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.

Adaptor Proteins, Vesicular Transport↗

PubMed1999

Serine phosphorylation and negative regulation of Stat3 by JNK.

STATs are activated by various cytokines and growth factors via tyrosine phosphorylation, which leads to sequential dimer formation, nuclear translocation, binding to specific DNA sequences, and regulation of gene expression. Recently, serine phosphorylation of Stat3 on Ser-727 by ERK has been identified in response to epidermal growth factor (EGF). Here, we report that Ser-727 phosphorylation of Stat3 can also be induced by JNK and activated either by stress or by its upstream kinase and that various stress treatments induce serine phosphorylation of Stat3 in the absence of tyrosine phosphorylation. Inhibitors of ERK and p38 did not inhibit UV-induced Stat3 serine phosphorylation, suggesting that neither of them is involved. We further demonstrate that JNK1, activated by its upstream kinase MKK7, negatively regulated the tyrosine phosphorylation and DNA binding and transcriptional activities of Stat3 stimulated by EGF. Correspondingly, pretreatment of cells with UV reduced the EGF-stimulated tyrosine phosphorylation and phosphotyrosine-dependent activities of Stat3. The inhibitory effect was not observed for Stat1. Our results suggest that Stat3 is a target of JNK that may regulate Stat3 activity via both Ser-727 phosphorylation-dependent and -independent mechanisms.

Amino Acid Substitution↗

PubMed1999

A helper virus-free packaging system for recombinant adeno-associated virus vectors.

Adeno-associated virus (AAV) is a human parvovirus that is currently receiving widespread attention for its potential use as a gene therapy vector. Construction of the recombinant AAV vector (rAAV) involves replacing most of the viral genome with a transgene of interest and then packaging this recombinant genome into an infectious virion. Most current protocols for generating rAAV entail the co-transfection of a vector plasmid and a packaging plasmid that expresses the viral replication and structural genes onto adenovirus (Ad) infected cells growing in culture. Limitations of this procedure include (1) contamination of rAAV with the Ad helper virus, (2) low yields of rAAV and (3) production of replication-competent AAV. In this report we describe new helper plasmids (pSH3 and pSH5) that eliminate the Ad co-infection requirement. The helper plasmids express the AAV rep and cap genes and the Ad E2A, VAI and E4 genes. When the helper plasmids are co-transfected onto human 293 cells with a vector plasmid in the absence of Ad infection, the rAAV vector yield is up to 80-fold greater than those obtained with the pAAV/Ad packaging plasmid. Moreover, replication competent AAV in the rAAV preparations is less than 0.00125%. The major advantages of this system are (1) the absence of infectious adenovirus and (2) the use of only two plasmids, which enhances transfection efficiencies and hence vector production. We believe that this two-plasmid transfection system will allow for more widespread use of the AAV vector system because of its simplicity and high yields. This system will be especially useful for preclinical analyses of multiple rAAV vectors.

Cell Line↗

PubMed1999

Separation and purification of isoflavones from Pueraria lobata by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied to the semipreparative separation and purification of puerarin and related isoflavones from a crude extract of Pueraria lobata. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v). Using the above solvent system the preparative HSCCC was successfully performed yielding six relatively pure isoflavones including puerarin from 80 mg of the crude extract in one-step separation.

Chromatography↗

PubMed1999

Protein kinase C delta associates with and phosphorylates Stat3 in an interleukin-6-dependent manner.

Stat3 is activated by phosphorylation on Tyr-705, which leads to dimer formation, nuclear translocation, and regulation of gene expression. Serine phosphorylation of Stat3 by mitogen-activated protein kinase has also been observed in cells responding to epidermal growth factor and shown to affect its tyrosine phosphorylation and transcriptional activity. Serine phosphorylation of Stat3 is also induced by interleukin-6 (IL-6) stimulation, which is shown to be independent of mitogen-activated protein kinase and sensitive to the Ser/Thr kinase inhibitor H7. In this study, we investigated whether protein kinase C (PKC) is the kinase that is induced and responsible for Stat3 serine phosphorylation by IL-6 stimulation and which isoform of PKCs is likely to be involved. Here, we report that Stat3 was specifically associated with PKC delta in vivo in an IL-6-dependent manner in several cell types. Furthermore, Stat3 was phosphorylated by PKC delta in vivo on Ser-727, which could be inhibited either by a specific PKC delta inhibitor or by a dominant-negative mutant of PKC delta. Finally, we showed that the phosphorylation of Stat3 by PKC delta led to a negative regulation of Stat3 DNA binding and transcriptional activity. These results indicate that PKC delta is likely to be the kinase that phosphorylates Stat3 in response to IL-6 stimulation and suggest a possible regulatory role of PKC delta on Stat3 function.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

Direct cloning of cell differential expression genes with full-length by a new strategy based on the multiple rounds of 'long distance' polymerase chain reaction and magnetic beads mediated subtraction.

The paper described a new cDNA subtractive cloning strategy. This strategy was based on the 'cap-finder' method, 'long distance' polymerase chain reaction (PCR), streptavidin magnetic beads mediated subtraction, and spin column chromatography. When PCR products were resolved on agarose gel after three rounds of subtraction, the 'single gene difference' group displayed a predominantly enriched band, but the 'multiple gene difference' group did not display any apparent difference. Of 200 clones inserted with 0.7-2 kb fragments from the 'multiple gene difference' group, 50% were identified to be new sequences and 35% were known. Of 100 new sequences, 35% contained coding regions and 75% were confirmed by dot-blotting to be differentially expressed by genes of the target cell. The results suggested that this strategy might be very efficient for full-length cloning of differentially expressed genes of the cell.

Cloning, Molecular↗

PubMed1999

Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex.

Traffic through the yeast Golgi complex depends on a member of the syntaxin family of SNARE proteins, Sed5p, present in early Golgi cisternae. Sft2p is a non-essential tetra-spanning membrane protein, found mostly in the late Golgi, that can suppress some sed5 alleles. We screened for mutations that show synthetic lethality with sft2 and found one that affects a previously uncharacterized membrane protein, Got1p, as well as new alleles of sed5 and vps3. Got1p is an evolutionarily conserved non-essential protein with a membrane topology similar to that of Sft2p. Immunofluorescence and subcellular fractionation indicate that it is present in early Golgi cisternae. got1 mutants, but not sft2 mutants, show a defect in an in vitro assay for ER-Golgi transport at a step after vesicle tethering to Golgi membranes. In vivo, inactivation of both Got1p and Sft2p results in phenotypes ascribable to a defect in endosome-Golgi traffic, while their complete removal results in an ER-Golgi transport defect. Thus the presence of either Got1p or Sft2p is required for vesicle fusion with the Golgi complex in vivo. We suggest that Got1p normally facilitates Sed5p-dependent fusion events, while Sft2p performs a related function in the late Golgi.

Alleles↗

PubMed1999

Enhanced antitumor effects of bone marrow transplantation in combination with fibroblast-mediated IL-2 and IL-3 gene therapy.

BACKGROUND: Bone marrow transplantation (BMT) and gene therapy are potent approaches to the recovery of bone marrow depression and induction of antitumor immunity after chemotherapy for the treatment of malignancies. In the present study, enhanced antitumor effect of BMT in combination with fibroblast-mediated interleukin (IL)-2 and IL-3 gene therapy was observed in tumor-bearing mice after chemotherapy. METHODS: BALB/c mice were inoculated s.c. with J558L plasmacytoma cells and injected i.p. with cyclophosphamide 300 mg/kg 3 days later. 24 hours after chemotherapy syngeneic bone marrow cells in combination with NIH3T3 fibroblast cells engineered to produce IL-2 (NIH3T3-IL-2) and/or NIH3T3 cells engineered to produce IL-3 (NIH3T3-IL-3) were implanted into the tumor-bearing mice. RESULTS: BMT in combination with implantation of either NIH3T3-IL-2 or NIH3T3-IL-3 cells exerted significant inhibition on the growth of J558L tumors and prolonged the survival period of the tumor-bearing mice as compared with the treatments with Hanks solution, BMT alone, or BMT plus implantation of NIH3T3 cells transduced with Neo gene. Synergistic antitumor effect was observed in mice after combined BMT and cytokine gene therapy. The cytotoxicities of natural killer cells, cytotoxic T lymphocytes, and macrophages in mice increased markedly after the combined treatment. Recovery of CFU-GM, CFU-MK and CFU-E formation in mice after combined therapy was accelerated obviously in mice after combined therapy. CONCLUSIONS: BMT in combination with fibroblast-mediated IL-2 and IL-3 gene therapy elicited augmented antitumor effects synergistically in tumor-bearing mice after chemotherapy mainly through induction of antitumor immune response and accelerated recovery of hematopoiesis.

3T3 Cells↗

PubMed1999

Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling.

Bone morphogenetic proteins (BMP) transduce their signals into the cell through a family of mediator proteins known as Smads. Upon phosphorylation by the BMP receptors, Smad1 interacts with Smad4 and translocates into the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription. However, the DNA-binding protein(s) involved in BMP signaling has not been identified. Using a yeast two-hybrid approach, we found that Smad1 interacts with Hoxc-8, a homeodomain transcription factor. The interaction between Smad1 and Hoxc-8 was confirmed by a "pull-down" assay and a co-immunoprecipitation experiment in COS-1 cells. Interestingly, purified Smad1 inhibited Hoxc-8 binding to the osteopontin Hoxc-8 site in a concentration-dependent manner. Transient transfection studies showed that native osteopontin promoter activity was elevated upon BMP stimulation. Consistent with the gel shift assay, overexpression of Hoxc-8 abolished the BMP stimulation. When a wild type or mutant Hoxc-8 binding element was linked to an SV40 promoter-driven reporter gene, the wild type but not the mutant Hoxc-8 binding site responded to BMP stimulation. Again, overexpression of Hoxc-8 suppressed the BMP-induced activity of the wild type reporter construct. Our findings suggest that Smad1 interaction with Hoxc-8 dislodges Hoxc-8 from its DNA binding element, resulting in the induction of gene expression.

Animals↗

PubMed1999

Enhanced therapeutic efficacy of tumor RNA-pulsed dendritic cells after genetic modification with lymphotactin.

Pulsing dendritic cells (DCs) with tumor cell-derived mRNA is regarded as an attractive alternative in the development of DC-based tumor vaccines. Our aim is to improve the therapeutic efficacy of DC-based tumor RNA vaccines by augmenting the preferential chemotaxis of DCs to T cells. Mouse bone marrow-derived DCs were genetically modified with lymphotactin (Lptn) by adenovirus vector, which conferred on DCs preferential chemotaxis to CD4+ and CD8+ T cells (Cao et al., 1998). Lptn gene-modified DCs (Lptn-DCs) were pulsed with tumor mRNA and used for vaccination in the tumor models of 3LL lung carcinoma and B16 melanoma. In both tumor models, immunization with 4 X 10(4) tumor RNA-pulsed Lptn-DCs induced more potent CTL activity, compared with their counterparts, specifically against tumor cells and Mut1 or tyrosinase-related protein 2 (TRP-2) peptide-pulsed RMA-S cells, and rendered the immunized mice resistant to tumor challenge much more effectively. CD8+ T cells were necessary and sufficient to generate the protection of Lptn-DC-based RNA tumor vaccines, and CD4+ T cells were required for the induction of tumor rejection. In the preestablished 3LL and B16 tumor models, vaccination with DC-based or LacZ-DC-based tumor RNA vaccines (2 X 10(5) cells) could reduce pulmonary metastasis and extend survival of tumor-bearing mice, but was less effective than the Lptn-DC counterpart (with 60-80% mice surviving). When the immunizing dose was decreased to 4 X 10(4) cells, Lptn-DC-based tumor vaccines rather than their counterparts were still significantly effective. Our studies provide a potential strategy to improve the efficacy of DC-based vaccines, and a new approach to immunological intervention by chemokines.

Adenoviridae↗

PubMed1999

Parathyroid hormone-regulated production of stem cell factor in human osteoblasts and osteoblast-like cells.

We investigated stem cell factor (SCF) expression in osteoblasts because mast cells, which occur ectopically in hyperparathyroid bone, are induced by SCF. Nontransformed osteoblasts and Saos2 or MG63 cells expressed SCF in response to PTH. Western analysis showed only large, cell-associated isoforms, Mrs approximately 40-48 kD. Transfection of MG63 cells with plasmids expressing antisense SCF mRNA eliminated immunoreactive SCF. Sequencing osteoblast SCF cDNAs showed that exon 6 was omitted. mRNAs without exon 6 produce membrane-associated SCF isoforms in rodents, suggesting that human SCFs are processed similarly. The major osteoblastic SCF mRNA, approximately 5 kB, was augmented by PTH. Neither protein or mRNA was increased by vitamin D, however, 6-7 kB transcripts were predominant in other tissues but not detectable in osteoblasts. We conclude that osteoblasts express SCF in response to PTH, with mRNA and protein processing differences relative to other cells. SCF stimulates osteoclasts, suggesting that PTH-induced osteoblastic SCF functions to accelerate bone turnover. Mast cells may occur due to SCF overexpression at extreme PTH levels.

Amino Acid Sequence↗

PubMed1999

Germline mutations are frequent in the APC gene but absent in the beta-catenin gene in familial adenomatous polyposis patients.

Inactivation of the adenomatous polyposis coli (APC) gene has been shown to initiate the majority of colorectal cancer (CRC), including a familial form called familial adenomatous polyposis (FAP). One consequence of the APC mutation is the activation of the beta-catenin (CTNNB1)/T-cell transcription factor (Tcf) pathway. A recent study has shown that about half of the sporadic CRC lacking APC mutation has CTNNB1 mutation, suggesting that CTNNB1 mutation can substitute for APC mutation in the initiation of colorectal tumorigenesis. However, the frequency of CTNNB1 germline mutation in FAP has not been reported. In the present study, we investigated the frequencies of APC and CTNNB1 germline mutations in 26 unrelated FAP families. We used the Protein Truncation Test (PTT) to screen the entire coding region of APC and found germline mutations in twenty families. We then screened for CTNNB1 germline mutations in the rest of the families lacking detectable APC mutations. No missense mutations at GSK-3beta phosphorylation sites or interstitial deletion of exon 3 of CTNNB1 was found. Our results indicate that APC germline mutations are frequent but CTNNB1 germline mutations are rare in FAP patients, suggesting that CTNNB1 mutation cannot substitute for APC mutation in the initiation of FAP. Genes Chromosomes Cancer 25:396-398, 1999.

Adenomatous Polyposis Coli↗

PubMed1999

pH-dependent conformational change of gastric mucin leads to sol-gel transition.

We present dynamic light scattering (DLS) and hydrophobic dye-binding data in an effort to elucidate a molecular mechanism for the ability of gastric mucin to form a gel at low pH, which is crucial to the barrier function of gastric mucus. DLS measurements of dilute mucin solutions were not indicative of intermolecular association, yet there was a steady fall in the measured diffusion coefficient with decreasing pH, suggesting an apparent increase in size. Taken together with the observed rise in depolarized scattering ratio with decreasing pH, these results suggest that gastric mucin undergoes a conformational change from a random coil at pH >/= 4 to an anisotropic, extended conformation at pH < 4. The increased binding of mucin to hydrophobic fluorescent with decreasing pH indicates that the change to an extended conformation is accompanied by exposure of hydrophobic binding sites. In concentrated mucin solutions, the structure factor S(q, t) derived from DLS measurements changed from a stretched exponential decay at pH 7 to a power-law decay at pH 2, which is characteristic of a sol-gel transition. We propose that the conformational change facilitates cross-links among mucin macromolecules through hydrophobic interactions at low pH, which in turn leads to a sol-gel transition when the mucin solution is sufficiently concentrated.

Animals↗

PubMed1999

Delivering neuroactive molecules from biodegradable microspheres for application in central nervous system disorders.

Nerve growth factor (NGF) may enhance axonal regeneration following injury to the central nervous system (CNS), such as after spinal cord injury. The release profile of NGF, co-encapsulated with ovalbumin, was tailored from biodegradable polymeric microspheres using both polymer degradation and protein loading. Biodegradable polymeric microspheres were prepared from PLGA 50/50, PLGA 85/15, PCL and a blend of PCL/PLGA 50/50 (1:1, w/w), where the latter was used to further tailor the degradation rate. The amount of protein loaded in the microspheres was varied, with PCL encapsulating the greatest amount of protein and PLGA 50/50 encapsulating the least. A two-phase release profile was observed for all polymers where the first phase resulted from release of surface proteins and the second phase resulted predominantly from polymer degradation. Polymer degradation influenced the release profile most notably from PLGA 50/50 and PLGA 85/15 microspheres. The amount and bioactivity of released NGF was followed over a 91 d period using a NGF-ELISA and PC12 cells, respectively. NGF was found to be bioactive for 91 d, which is longer than previously reported.

Animals↗

PubMed1999

Cyclic compression of articular cartilage explants is associated with progressive consolidation and altered expression pattern of extracellular matrix proteins.

In this study, we investigated the biosynthetic response of full thickness, adult bovine articular cartilage explants to 45 h of static and cyclic unconfined compression. The cyclic compression of articular cartilage resulted in a progressive consolidation of the cartilage matrix. The oscillatory loading increased protein synthesis ([35S]methionine incorporation) by as much as 50% above free swelling control values, but had an inhibitory influence on proteoglycan synthesis ([35SO4] incorporation). As expected, static compression was associated with a dose-dependent decrease in biosynthetic activity. ECM oligomeric proteins which were most affected by mechanical loading were fibronectin and cartilage oligomeric matrix protein (COMP). Static compression at all amplitudes caused a significant increase in fibronectin synthesis over free swelling control levels. Cyclic compression of articular cartilage at 0.1 Hz and higher was consistently associated with a dramatic increase in the synthesis of COMP as well as fibronectin. The biosynthetic activity of chondrocytes appears to be sensitive to both the frequency and amplitude of the applied load. The results of this study support the hypothesis that cartilage tissue can remodel its extracellular matrix in response to alterations in functional demand.

Animals↗

PubMed1999

Therapy of established tumour with a hybrid cellular vaccine generated by using granulocyte-macrophage colony-stimulating factor genetically modified dendritic cells.

Dendritic cells (DCs) are the most powerful of all antigen-presenting cells and play a critical role in the induction of primary immune responses. DC-based vaccination represents a potentially powerful strategy for cancer immunotherapy. In this study, a new approach for a DC-based melanoma vaccine was described. Splenic DCs from C57BL/6 mice were fused with B16 melanoma cells, and the resultant B16/DC hybrid cells expressed major histocompatibility complex (MHC) molecules - B7 as well as the B16 tumour marker M562 - which were enriched by Ia-mediated positive selection with a MiniMACS column. The fusion rates were 12.7-26.8%. To generate hybrid tumour vaccines with potentially greater potent therapeutic efficacy, we genetically engineered DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) prior to cell fusion. Recombinant adenovirus vector was used to mediate gene transfer into DCs with high efficiency and DCs expressed GM-CSF at 96-138 ng/105 cells/ml 24 hr after GM-CSF gene transfer. GM-CSF gene-modified DCs (DC.GM) exhibited higher expression of B7 and co-stimulatory capacity in mixed lymphocyte reaction (MLR). Fusion of DC.GM with B16 cells generated B16/DC.GM hybrid cells secreting GM-CSF at 59-63 ng/105 cells/ml. Immunization of C57BL/6 mice with the B16/DC hybrid vaccine elicited a specific cytotoxic T-lymphocyte (CTL) response and protected the immunized mice from B16 tumour challenge, reduced pulmonary metastases and extended the survival of B16 tumour-bearing mice. The B16/DC.GM hybrid vaccine was able to induce a CTL response and protective immunity more potently and tended to be therapeutically more efficacious than the B16/DC vaccine. In vivo depletion of T-cell subsets demonstrated that both CD8+ and CD4+ T cells were essential for the therapeutic effects of B16/DC and B16/DC.GM hybrid vaccines. Additionally, other non-specific effector cells may also contribute to tumour rejection induced by the B16/DC.GM hybrid vaccine. These data indicate that a DC-based hybrid tumour vaccine may be an attractive strategy for cancer immunotherapy, and that GM-CSF gene-modified DCs may lead to the generation of hybrid vaccines with potentially increased therapeutic efficacy.

Animals↗