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Biomedical subjects

X Feng

Publications and source records attributed to X Feng.

At least 73 records · Page 4Linked to original sources
PubMed2000

Transforming growth factor-beta up-regulates the beta 5 integrin subunit expression via Sp1 and Smad signaling.

Integrin-mediated cell-matrix interactions play important roles in regulating cell function. Since transforming growth factor-beta (TGF-beta) modulates many osteoblast activities, we hypothesized that the growth factor acts in part by modulating integrin expression. TGF-beta increased cell adhesion to vitronectin and up-regulated the surface level of alpha(v)beta(5) via increasing beta(5) protein synthesis by a transcriptional mechanism. Promoter activity analysis demonstrated that a TGF-beta-responsive element resides between nucleotides -63 and -44. Electrophoretic mobility shift assay and immunoprecipitation/Western studies indicated that the nuclear complex formed using the -66/-42 oligonucleotide contained both Sp1/Sp3 and Smad proteins. Since nuclear Sp1/Sp3 levels were not altered, whereas Smad levels were increased by TGF-beta, we investigated the roles of Smad proteins in the up-regulation of beta(5) gene activation. Co-transfection of cells with beta(5) promoter reporter construct and expression vectors for Smad3, Smad4, and Sp1 increased the stimulatory effect of TGF-beta. Furthermore, expression of dominant negative Smad3 or Smad4 in cells decreased or abolished the stimulation of beta(5) promoter activity by TGF-beta. Smad4 mutant also inhibited the up-regulation of surface beta(5) level by TGF-beta. Thus, TGF-beta increases expression of the integrin beta(5) gene by mechanisms involving Sp1/Sp3 and Smad transcription factors.

Animals↗
PubMed2000

CD8+ T cells are capable of rejecting pancreatic islet xenografts.

BACKGROUND: In this study, the capacity of CD8+ T cells to act as a potential effector mechanism in pancreatic xenograft rejection was examined. METHODS: The fate of pancreatic islet xenografts was studied in mice deficient in MHC class II molecules and CD4+ T cells. Fetal pig pancreas (FPP) or Wistar rat islets (RI) were transplanted into nondiabetic or streptozotocin-induced diabetic I-A knock-out (CII K/O) mice. RESULTS: CII K/O mice were capable of rejecting both RI and FPP grafts. RI graft survival was not prolonged compared with wild type C57BL/6 controls. However, FPP grafts did survive longer in CII K/O recipients than in C57BL/J6 mice. Both RI and FPP graft rejection were CD8+ T-cell phenomena in CII K/O mice, as anti-CD8 monoclonal antibody prolonged graft survival, there were increased CD8+ T cells in the grafts and spleens of CII K/O recipients, and cell-mediated cytotoxicity was a CD8+ T-cell phenomenon associated with activation of the perforin/granzyme B system. By contrast, RI and FPP graft rejection was a CD4+ T cell-dependent phenomenon in wild type C57BL/6 mice with graft survival prolonged by anti-CD4 monoclonal antibody. There were increased numbers of CD4+ T cells, and cell-mediated cytotoxicity was a CD4+ T-cell phenomenon associated with activation of the Fas/FasL lytic pathway. CONCLUSIONS: The results demonstrate that, in the absence of CD4+ T cells, CD8+ T cells were capable of rejecting both rat and pig pancreatic islet xenografts.

Animals↗
PubMed2000

Regulation of receptor-mediated protein kinase C membrane trafficking by autophosphorylation.

Signal transduction via protein kinase C (PKC) is closely regulated by its subcellular localization. In response to activation of cell-surface receptors, PKC is directed to the plasma membrane by two membrane-targeting domains, namely the C1 and C2 regions. This is followed by the return of the enzyme to the cytoplasm, a process shown recently to require PKC autophosphorylation (Feng, X., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 26870-26874). In the present study, we examined mechanisms of translocation and reverse translocation and the role of autophosphorylation in these processes. By visualizing the trafficking of wild-type as well as mutant PKCbetaII in live cells, we demonstrated that in response to cell-surface receptor activation, the function of the C1 region is required but not sufficient for recruitment of the enzyme to the plasma membrane. The C2 region is also critical in anchoring the enzyme to the plasma membrane. Furthermore, the inability of a kinase-deficient PKC to undergo reverse translocation was restored by the addition of intracellular calcium chelators, suggesting a role for the C2 region in the persistent phase of translocation. On the other hand, the inability of a C2 deletion mutant (C1 region intact) to translocate in response to agonist was reversed in mutants lacking kinase activity or by mutation of the Ser(660) autophosphorylation site to alanine, suggesting that autophosphorylation of this site is required for opposing the action of the C2 region. Therefore, the membrane-targeting function of the C1 region is facilitated by the C2 region and appears to be opposed by autophosphorylation. Taken together, these findings provide novel evidence of the functional regulation of reversible PKC membrane localization by autophosphorylation, and they show that the dynamic translocation of PKC in response to agonists is tightly regulated in a collaborative fashion by the C1 and C2 regions in balance with the effects of autophosphorylation.

Biological Transport↗
PubMed2000

Concentration-dependent dual effect of thrombin on impaired growth/apoptosis or mitogenesis in tumor cells.

Because thrombin-treated tumor cell-induced metastasis increases tumor nodule volume(12) greater than nodule number, we studied the effect of thrombin on tumor cell growth in vitro and in vivo (murine B16F10 melanoma, human HCT8 colon carcinoma, DU145 prostate carcinoma). Tumor cell growth was measured after 3 to 7 days in 1% fetal calf serum (FCS) + RPMI 1640. We found that, whereas relatively low concentrations of thrombin, 0.1 to 0.5 U/mL (1-5 nmol/L) enhance tumor cell growth in vitro approximately 2- to 3-fold, higher concentrations, 0.5 to 1 U/mL (5-10 nmol/L) impaired cell growth approximately 2- to 4-fold. Impaired cell growth was associated with cell cycle arrest at G(2)M and increased pre-G(o) DNA, as well as apoptosis, measured by tumor cell binding to Annexin V and propidium iodide. Apoptosis was reversed with the general caspase inhibitor, FK-011. The enhancing and inhibiting effects were specific for thrombin (reversed with inactive diisopropyl-fluorophosphate [DFP]-thrombin) and mediated via the protease-activated receptor 1 (PAR-1). PAR-1 activation was demonstrated by (1) use of a cell line, B16F10, devoid of the 3 other thrombin receptors, PAR-3, PAR-4, and GPIb; and (2) greater sensitivity of PAR-1 transfected B16F10 and HCT8 cells to impaired cell growth/apoptosis, 3- and 14-fold, respectively. Thus, thrombin has a bimodal effect on PAR-1 in tumor cells: enhanced growth at low concentration, impaired growth/apoptosis at higher concentration.

Animals↗
PubMed2000

Characterization of the functionally related sites in the neural inducing gene noggin.

Previously we have shown that blocking bone morphogenetic protein (BMP) receptor signaling by a dominant negative BMP receptor causes neurogenesis in Xenopus animal caps (ACs), whereas the physiological neural inducer noggin acts as a homodimer physically binding to BMP-4 and disrupting its signaling at the ligand level. The present study attempted to elucidate the relationship between the structure and function of noggin. By replacing some cysteine residues with serine residues through a site-directed mutagenesis strategy, we generated three noggin mutants, C145S, C205S, and C(218, 220, 222)S (3CS). Although mRNAs encoded by these mutants were translated as efficiently as wild-type (WT) noggin mRNA, they behaved differently when expressed in vivo. Expression of WT noggin or C205S in Xenopus ACs converted the explants (prospective ectoderm) into neural tissue, indicated by the neural-like morphology and expression of the pan neural marker NCAM in the ACs. In contrast, ACs expressing C145S or 3CS sustained an epidermal fate like the control caps. Similar results were observed in the mesoderm where C205S (but not C145S and 3CS) displayed dorsalizing activity as well as WT noggin. Altogether, our results suggest that Cys145 alone or Cys(218, 220, 222) as a whole in noggin protein is required for the biological activities of noggin, probably participating in the dimerization of noggin with BMP-4 or itself.

Animals↗
PubMed2000

Porphyrazinediols: Synthesis, Characterization, and Complexation to Group IVB Metallocenes.

The first metalated porphyrazinediols 11 have been prepared from (L)-(+)-dimethyl tartrate via conversion into the corresponding dispoke or 2,3-dimethoxy-2,3-butanediyl protected 2,3-dihydroxymaleonitrile, Linstead macrocyclization, transmetalation, and deprotection. Their stability is very dependent on the nature of the metal ion in the cavity of the porphyrazine. Reaction of these porphyrazinediols with metallocene dichlorides led to new solitaire porphyrazines 12 while DDQ oxidation followed by trapping with diaminomaleonitrile afforded new porphyrazine dinitriles 14.

Journal Article↗
PubMed2000

Sp1/Sp3 and PU.1 differentially regulate beta(5) integrin gene expression in macrophages and osteoblasts.

Murine osteoclast precursors and osteoblasts express the integrin alpha(v)beta(5), the appearance of which on the cell surface is controlled by the beta(5), and not the alpha(v), subunit. Here, we show that a 173-base pair proximal region of the beta(5) promoter mediates beta(5) basal transcription in macrophage (osteoclast precursor)-like and osteoblast-like cells. DNase I footprinting reveal four regions (FP1-FP4) within the 173-base pair region, protected by macrophage nuclear extracts. In contrast, osteoblast nuclear extracts protect only FP1, FP2, and FP3. FP1, FP2, and FP3 bind Sp1 and Sp3 from both macrophage and osteoblast nuclear extracts. FP4 does not bind osteoblast proteins but binds PU.1 from macrophages. Transfection studies show that FP1 and FP2 Sp1/Sp3 sites act as enhancers in both MC3T3-E1 (osteoblast-like) and J774 (macrophage-like) cell lines, whereas the FP3 Sp1/Sp3 site serves as a silencer. Mutation of the FP2 Sp1/Sp3 site totally abolishes promoter activity in J774 cells, with only partial reduction in MC3T3-E1 cells. Finally, we demonstrate that PU.1 acts as a beta(5) silencer in J774 cells but plays no role in MC3T3-E1 cells. Thus, three Sp1/Sp3 sites regulate beta(5) gene expression in macrophages and osteoblast-like cells, with each element exhibiting cell-type and/or activation-suppression specificity.

Animals↗
PubMed2000

Fractal stream chemistry and its implications for contaminant transport in catchments

The time it takes for rainfall to travel through a catchment and reach the stream is a fundamental hydraulic parameter that controls the retention of soluble contaminants and thus the downstream consequences of pollution episodes. Catchments with short flushing times will deliver brief, intense contaminant pulses to downstream waters, whereas catchments with longer flushing times will deliver less intense but more sustained contaminant fluxes. Here we analyse detailed time series of chloride, a natural tracer, in both rainfall and runoff from headwater catchments at Plynlimon, Wales. We show that, although the chloride concentrations in rainfall have a white noise spectrum, the chloride concentrations in streamflow exhibit fractal 1/f scaling over three orders of magnitude. The fractal fluctuations in tracer concentrations indicate that these catchments do not have characteristic flushing times. Instead, their travel times follow an approximate power-law distribution implying that they will retain a long chemical memory of past inputs. Contaminants will initially be flushed rapidly, but then low-level contamination will be delivered to streams for a surprisingly long time.

Journal Article↗
PubMed2000

Stable isotopes examined across a migratory divide in Scandinavian willow warblers (Phylloscopus trochilus trochilus and Phylloscopus trochilus acredula) reflect their African winter quarters.

The C and N isotopes of feathers from two subspecies of willow warblers Phylloscopus trochilus trochilus and Phylloscopus trochilus acredula) are isotopically distinct. Our analysis of 138 adult males from 14 sites distributed across Sweden shows that the mean delta15N and delta13C values of subspecies acredula (from latitudes above 63 degrees N) were significantly higher than the mean delta15N and delta13C values of subspecies trochilus (from latitudes below 61 degrees N). The analysed willow warbler feathers had been moulted in the winter quarters and the observed isotopic signatures should thus reflect the isotopic pattern of food assimilated in Africa. The isotopic data observed in Sweden match the cline in morphology, both showing abrupt changes around 62 degrees N. This result agrees with data from ringing recoveries indicating that the two subspecies occupy geographically and isotopically distinct wintering grounds in Africa. Our isotopic data suggest that analysis of stable isotopes of C and N is a promising method to track wintering quarters of European birds that migrate to Africa.

Africa↗
PubMed2000

Regulation of alphaVbeta3 and alphaVbeta5 integrins by dexamethasone in normal human osteoblastic cells.

Long-term administration of pharmacological doses of glucocorticoids inhibits bone formation and results in osteoporosis. Since integrin-mediated cell-matrix interactions are essential for osteoblast function, we hypothesized that the detrimental effect of glucocorticoids on bone derived, at least in part, from decreased integrin-matrix interactions. Because alphavbeta3 and alphavbeta5 integrins can interact with several bone matrix proteins, we analyzed the effects of dexamethasone (Dex) on the expression of these integrins in normal human osteoblastic cells. We found adhesion of these cells to osteopontin and vitronectin to be dependent on alphavbeta3 and alphavbeta5, respectively; this ligand specificity was not altered by Dex. The effects of Dex on the adhesion of human osteoblastic cells to osteopontin and vitronectin were biphasic with an increase after 2 days, followed by a decrease after 8 days of treatment. Consistently, surface alphavbeta3 and alphavbeta5 integrins, which were increased after 2 days of Dex treatment, were decreased after 8 days. Similarly, total cellular alphav, beta3, and beta5 proteins, which were increased by Dex early in the culture, were diminished after 8 days. Metabolic labeling studies indicated that Dex exhibited biphasic regulation on the biosynthesis of alphavbeta5, with stimulation observed during the second day of treatment, followed by inhibition during the 8th day of exposure. By contrast, the biosynthesis of alphavbeta3 was inhibited by Dex on day 1 and remained inhibited on day 8. Analysis of the mRNA indicated that alphav and beta5 levels were increased by Dex during early exposure (1-3 days), followed by inhibition after prolonged exposure (>/=7 days). By contrast, Dex decreased beta3 mRNA level at all the time points analyzed. Consistently, Dex decreased beta3 promoter activity after 1 day and persisted over 8-day period. By contrast, Dex stimulated beta5 promoter activity after 1 or 2 days but had no effect after 8 days. To further evaluate mechanism(s) leading to the decreased integrin expression after prolonged Dex treatment, mRNA stability was analyzed. Dex was found to accelerate the degradation of alphav, beta3 and beta5 mRNA after an 8-day treatment. Thus, the regulation of alphavbeta3 was dependent on transcription and posttranscriptional events whereas the expression of alphavbeta5 was dependent mainly on posttranscriptional events after prolonged Dex treatment. In conclusion, Dex exhibited time-dependent regulation on the expression of alphavbeta3 and alphavbeta5 integrins in normal human osteoblastic cells. Short-term exposure to Dex increased the levels of alphavbeta3 and alphavbeta5 on the surface and cell adhesion to osteopontin and vitronectin whereas long-term exposure to Dex decreased the expression of both integrins and inhibited the cell adhesion to matrix proteins.

Antigens, CD↗
PubMed2000

Modified on-line monitoring of total gaseous mercury in flue gases using Semtech Hg 2000 analyzer.

The Semtech Hg 2000 analyzer continuously monitors the Hg0 content in flue gas. An on-line measurement method of total gaseous mercury in flue gas developed in our laboratory is described, which uses the absorption cell of the Semtech Hg Analyzer connected to a converter that is located in a furnace heated up to 650 degrees C. The converter can be heated up to 800 degrees C by both the furnace and an extra heating of a Ni-Cr alloy heating wire. Both the absorption cell and the converter are made of quartz. All gaseous Hg2+ species in flue gas are thermally reduced to Hg0 by the converter and detected by the Semtech Hg 2000 analyzer. The thermal reduction efficiencies of different conversion materials, which were filled in the converter, such as quartz chips, granular MgO, Ni and CoO powder, were tested using different flue gas conditions. Studies have shown that HCI is the major factor to inhibit the thermal reduction of Hg2+ to Hg0, and in the converter and the absorption cell Hg0 will react readily with HCl to form HgCl2. Both MgO and Ni could be used in the converter to absorb HCl in the flue gas, but Ni has better absorption efficiency. By using an original Semtech and a modified one, both Hg0 and total gaseous Hg contents in flue gas could be monitored simultaneously and continuously.

Air Pollutants↗
PubMed2000

Improved determination of gaseous divalent mercury in ambient air using KCl coated denuders.

An improved method for the determination of gaseous divalent mercury (GDM) in ambient air using KCl coated denuders has been developed and tested. GDM collected in the KCl coated denuders can be quantitatively desorbed at 450 degrees C in 10 min. After being complete thermally reduced to Hg0 at 900 degrees C, all mercury released from the denuder is pre-concentrated on the analytical Au trap, and detected by cold vapor atomic fluorescence spectrometry (CVAFS). The absolute detection limit of the method is less than 3 pg. Preliminary data of GDM concentration in ambient air from different sampling stations show that GDM concentrations in the urban air of Göteborg are much higher than in rural air (Rörvik and Sasetta), which indicates the anthropogenic origin of GDM.

Journal Article↗
PubMed2000

Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum.

Human cryptosporidiosis is attributed to two major Cryptosporidium parvum genotypes of which type 1 appears to be the predominant. Most laboratory investigations however are performed using genotype 2 isolates, the only type which readily infects laboratory animals. So far type 1 has only been identified in humans and primates. A type 1 isolate, obtained from an individual with HIV and cryptosporidiosis, was successfully adapted to propagate in gnotobiotic piglets. Genotypic characterization of oocyst DNA from this isolate using multiple restriction fragment length polymorphisms, a genotype-specific PCR marker, and direct sequence analysis of two polymorphic loci confirmed that this isolate, designated NEMC1, is indeed type 1. No changes in the genetic profile were identified during multiple passages in piglets. In contrast, the time period between infection and onset of fecal oocyst shedding, an indicator of adaptation, decreased with increasing number of passages. Consistent with other type 1 isolates, NEMC1 failed to infect mice. A preliminary survey of the NEMC1 genome covering approximately 2% of the genome and encompassing 200 kb of unique sequence showed an average similarity of approximately 95% between type 1 and 2 sequences. Twenty-four percent of the NEMC1 sequences were homologous to previously determined genotype 2 C. parvum sequences. To our knowledge, this is the first successful serial propagation of genotype 1 in animals, which should facilitate characterization of the unique features of this human pathogen.

Animals↗
PubMed2000

Single isolated droplets with net charge as a source of ions

A levitation device for charged particles has been used to position a single isolated droplet at a time at atmospheric pressure near the sampling orifice of a vacuum chamber. Following a brief desolvation period (550 ms), a series of coluomb fission events were initiated. Several atmospheric pressure electrode designs were evaluated with respect to guiding the progeny droplets/ions, to the sampling orifice. The best design tested consisted of a series of four annular electrodes of decreasing radius positioned above the levitation ring electrodes, and, on average, 40 ions were counted per single isolated droplet. The ion utilization (charge detected versus charge in the original droplet) with this electrode design has been estimated to be 5 x 10(-6), a substantial improvement over the current utilization measured to be < or = 1 x 10(-9) with a conventional electrospray ion source using the same vacuum apparatus.

Journal Article↗
PubMed2000

Determination of a molecular torsional angle in the metarhodopsin-I photointermediate of rhodopsin by double-quantum solid-state NMR.

We present a solid-state NMR study of metarhodopsin-1, the pre-discharge intermediate of the photochemical signal transduction cascade of rhodopsin, which is the 41 kDa integral membrane protein that triggers phototransduction in vertebrate rod cells. The H-C10-C11-H torsional angles of the retinylidene chromophore in bovine rhodopsin and metarhodopsin-I were determined simultaneously in the photo-activated membrane-bound state, using double-quantum heteronuclear local field spectroscopy. The torsional angles were estimated to be [phi] = 160+/-10 degrees for rhodopsin and phi = 180+/-25 degrees for metarhodopsin-I. The result is consistent with current models of the photo-induced conformational transitions in the chromophore, in which the 11-Z retinal ground state is twisted, while the later photointermediates have a planar all-E conformation.

Animals↗