Biomedical subjects

C S Chen

Publications and source records attributed to C S Chen.

At least 91 records · Page 5Linked to original sources

PubMed1998

Further characterization of a new in vitro angiogenesis model under serum free culture conditions; suppression of endothelial cell differentiation by serum.

We studied the regulation of the extracellular matrix in the recently established murine vascular endothelial cell clones, F-2 or F-2C. F-2 cells constitutively show a cobblestone growth pattern under serum supplemented culture conditions, whereas F-2C cells undergo spontaneous histodifferentiation to form tubular structures in chemically defined media. We reported that the tubulogenesis induced by F-2C might relate to the heavy deposition of a 'basement membrane analog' as a subendothelial matrix (SEM). We further characterized the regulation of extracellular matrix (ECM) metabolism in these cell clones, in terms of gelatinase expression, ECM degradation and the effects of serum. F-2C cells in culture medium containing 1% serum did not undergo tubulogenesis but presented cobblestone growth. Zymography analysis showed that both F-2 and F-2C cells express two gelatinase (72 and 92 kDa). However, F-2 cells mainly expressed the former and faintly the latter, whereas F-2C mainly expressed the latter. Proteolysis studies showed that the spent media conditioned by F-2C cells partially cleaved type IV collagen and completely degraded type V collagen. The cleavage of type V collagen was suppressed by the addition of serum, whereas that of type IV collagen was not. The proteolysis of laminin and fibronectin by the conditioned medium was not observed. Serum-supplemented F-2 or F-2C cultures markedly suppressed SEM deposition. These results indicated that F-2C cells under serum free culture conditions not only present a simple and useful in vitro model with which to study the dynamic processes of proteolysis and ECM metabolism during the sequential phases of angiogenesis, but is also useful for analyzing the serum effects on angiogenesis (AG).

Animals↗

PubMed1998

Cellular control lies in the balance of forces.

Mechanical tension generated within the cytoskeleton of living cells is emerging as a critical regulator of biological function in diverse situations ranging from the control of chromosome movement to the morphogenesis of the vertebrate brain. In this article, we review recent advances that have been made in terms of understanding how cells generate, transmit and sense mechanical tension, as well as how they use these forces to control their shape and behavior. An integrated view of cell regulation that incorporates mechanics and structure as well as chemistry is beginning to emerge.

Animals↗

PubMed1998

Monitoring soluble interleukin-2 receptor levels in related and unrelated donor allogenic bone marrow transplantation.

Acute graft-versus-host disease (GVHD) is effected by donor T lymphocytes which have been stimulated by host antigens. Activated donor T lymphocytes express interleukin-2 receptor (IL-2R), which is comprised of three subunits (alpha, beta, gamma). During activation, the a IL-2R subunit (CD25) is shed from the receptor complex and can be measured in the circulation. Soluble IL-2Ralpha (sIL-2R) levels are increased in states of immune activation including GVHD, and could theoretically be used as a guide to therapy. Since IL-2Ralpha expression is an early marker of T cell activation, we investigated: (1) if an increase in sIL-2R is specific for acute GVHD; and (2) if serial sIL-2R levels can identify patients with early GVHD, prior to the onset of clinical tissue damage (effector function). Weekly sIL-2R levels were monitored in 36 patients undergoing matched related (n=23) or matched unrelated (n=13) allogeneic bone marrow transplantation (BMT). There was no significant difference in sIL-2R levels between matched related and matched unrelated recipients. Patients with acute GVHD (n=19, 53%) demonstrated higher sIL-2R levels, than those without during weeks 2 and 3 post-BMT (P=0.02 and 0.04, Mann-Whitney U test, two-tailed). In patients with acute GVHD, the rise in sIL-2R preceded the clinical signs of GVHD (16/19 patients). However, patients with sepsis demonstrated a trend towards higher sIL-2R levels at week 1 and significantly greater levels by week 4 (P=0.02). Furthermore, patients with veno-occlusive disease (VOD) (25%) also had significantly higher sIL-2R levels at week 2 (P=0.03). We conclude that although sIL-2R levels increase in patients with acute GVHD, similar increases are seen in patients with VOD and/or sepsis and therefore, as a single biochemical marker, we find that serial measurements of sIL-2R lacks sufficient specificity to guide GVHD therapy.

Acute Disease↗

PubMed1998

Control of cyclin D1, p27(Kip1), and cell cycle progression in human capillary endothelial cells by cell shape and cytoskeletal tension.

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27(Kip1), and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.

Actins↗

PubMed1998

Case report: Spontaneous isolated mesenteric fibromatosis presenting as megaduodenum.

A case of spontaneous isolated mesenteric fibromatosis presenting as megaduodenum is reported. The lesion was small, only 2 cm in its greatest diameter. However, because of its location near the ligament of Treitz and because of its characteristic infiltrative growth pattern, the tumour involved the distal duodenum and resulted in acute angulation and obstruction early in its course. We successfully resected the mass and the patient is in fair physical condition 6 months after operation.

Adult↗

PubMed1998

Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation.

The antibacterial effects of sulfonated and sulfobenzoyl chitosans were evaluated and compared with that of 69% deacetylated chitosan (DD69 chitosan). Minimal inhibitory concentrations of sulfonated chitosan (SC1, 0.63% sulfur content) against Shigella dysenteriae, Aeromonas hydrophila, Salmonella typhimurium, and Bacillus cereus were found to be lower than those of DD69 chitosan. A high sulfur content in sulfonated chitosan adversely influenced its antibacterial effect. Sulfobenzoyl chitosan (SBC) has excellent water solubility and an antibacterial effect comparable to that of SC1. SBC at 1,000 and 2,000 ppm extended the shelf life of oysters at 5 degrees C by 4 days at the former or by 7 days at least at the latter concentration. The growth of coliforms and Pseudomonas, Aeromonas, and Vibrio species on oysters was retarded by the addition of DD69 chitosan or SBC.

Animals↗

PubMed1998

Influences of walking speed change on the lumbosacral joint force distribution.

To more understand the influence of the walking speed on the spinal joint force distribution, a three-dimensional biomechanical model was used to estimate the spine loads during human gait with three different walking speeds. This previously developed and validated model included a dynamic external model and an internal model with forces of disc, 8 major muscles, 2 ligaments and 2 facet joints at L5/S1 level. A linear optimization method was used to solve the internal model to estimate the L5/S1 spinal joint force distribution. The results of five young male subjects showed that the mean peak L5/S1 disc compressive forces on the slow, preferred and fast speeds were 2.28, 2.53, 2.95 body weight, respectively. The peak forces happened right after the heel strike and before completely toe off. The facet joint forces were generally increased with the walking speed increase, too. To reduce the loads on the spine, the slow walking is then recommended for the patients with low back pain or after spinal surgery.

Acceleration↗

PubMed1998

Dideoxy fingerprinting for rapid screening of rpoB gene mutations in clinical isolates of Mycobacterium tuberculosis.

Rifampin is a key component of therapeutic regimens for tuberculosis control, and a marker for multidrug resistance of Mycobacterium tuberculosis. Mutations responsible for conferring rifampin resistance in M. tuberculosis are known to occur in a 69-bp region of the rpoB gene. In this study, we assessed the accuracy of dideoxy fingerprinting (ddF), a hybrid technique employing elements of dideoxy sequencing and single-strand polymorphism analysis, for rapid screening of rifampin resistance in clinical isolates of M. tuberculosis. This technique was used to analyze 72 M. tuberculosis isolates. The results were compared with those of automated dideoxy sequencing and the antibiotic resistance profile (determined with the BACTEC system). Of the 72 isolates, 50 were rifampin resistant. The ddF findings were completely consistent with those of dideoxy sequencing in all isolates. In 68 (94%) isolates, the ddF findings were consistent with the rifampin resistance status determined with the BACTEC system; all four isolates with inconsistent results had no mutation in the 69-bp region, but were resistant to rifampin. Our findings suggest that ddF accurately detects mutations in the rifampin resistance-associated 69-bp region of the rpoB gene in clinical isolates of M. tuberculosis, and may be a valuable screening tool for rifampin resistance.

DNA Fingerprinting↗

PubMed1997

A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains.

Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (PtdIns)-4-P and PtdIns-4,5-P2 and have not considered the lipid products of phosphoinositide 3-kinase: PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to PtdIns-3,4, 5-P3 with higher affinity than to PtdIns-4,5-P2, PtdIns-3,4-P2 or inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4). This selectivity is decreased by the xid mutation (R28C). Selective binding of PtdIns-3,4,5-P3 over PtdIns-4,5-P2 or PtdIns-3,4-P2 was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the beta-adrenergic receptor kinase. The oxysterol binding protein and beta-spectrin PH domains bound PtdIns-3,4,5-P3 and PtdIns-4,5-P2 with similar affinities. PtdIns-3,4,5-P3 and PtdIns-4,5-P2 also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1997

Gelsolin binding to phosphatidylinositol 4,5-bisphosphate is modulated by calcium and pH.

The actin cytoskeleton of nonmuscle cells undergoes extensive remodeling during agonist stimulation. Lamellipodial extension is initiated by uncapping of actin nuclei at the cortical cytoplasm to allow filament elongation. Many actin filament capping proteins are regulated by phosphatidylinositol 4,5-bisphosphate (PIP2), which is hydrolyzed by phospholipase C. It is hypothesized that PIP2 dissociates capping proteins from filament ends to promote actin assembly. However, since actin polymerization often occurs at a time when PIP2 concentration is decreased rather than increased, capping protein interactions with PIP2 may not be regulated solely by the bulk PIP2 concentration. We present evidence that PIP2 binding to the gelsolin family of capping proteins is enhanced by Ca2+. Binding was examined by equilibrium and nonequilibrium gel filtration and by monitoring intrinsic tryptophan fluorescence. Gelsolin and CapG affinity for PIP2 were increased 8- and 4-fold, respectively, by microM Ca2+, and the Ca2+ requirement was reduced by lowering the pH from 7.5 to 7.0. Studies with the NH2- and COOH-terminal halves of gelsolin showed that PIP2 binding occurred primarily at the NH2-terminal half, and Ca2+ exposed its PIP2 binding sites through a change in the COOH-terminal half. Mild acidification promotes PIP2 binding by directly affecting the NH2-terminal sites. Our findings can explain increased PIP2-induced uncapping even as the PIP2 concentration drops during cell activation. The change in gelsolin family PIP2 binding affinity during cell activation can impact divergent PIP2-dependent processes by altering PIP2 availability. Cross-talk between these proteins provides a multilayered mechanism for positive and negative modulation of signal transduction from the plasma membrane to the cytoskeleton.

Calcium↗

PubMed1997

Geometric control of cell life and death.

Human and bovine capillary endothelial cells were switched from growth to apoptosis by using micropatterned substrates that contained extracellular matrix-coated adhesive islands of decreasing size to progressively restrict cell extension. Cell spreading also was varied while maintaining the total cell-matrix contact area constant by changing the spacing between multiple focal adhesion-sized islands. Cell shape was found to govern whether individual cells grow or die, regardless of the type of matrix protein or antibody to integrin used to mediate adhesion. Local geometric control of cell growth and viability may therefore represent a fundamental mechanism for developmental regulation within the tissue microenvironment.

Animals↗

PubMed1997

Role of modified glutamic acid in the helical structure of conantokin-T.

Circular dichroism (CD) and 2-dimensional NMR were used to study the solution conformation of conantokin-T (Con-T), a small peptide toxin found in the venom of fish-hunting cone snails, and its Glu-substituted analog. Con-T lacks disulfide bonds but contains many gamma-carboxyglutamic acids (Gla), a post-translationally modified residue. Our results show that Con-T adopts an alpha-helical conformation in aqueous solution even in the absence of calcium. Glu replacements diminish both helicity and function of Con-T. The helical content of Con-T is higher than most natural helical peptides of this length in aqueous solution. The sequence of this small toxin incorporates several known elements that stabilize alpha-helical structure in peptides. Gla residues form several salt bridges that stabilize helical conformation of Con-T.

1-Carboxyglutamic Acid↗

PubMed1997

The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C.

Phosphoinositide 3-kinase has been implicated as an activator of cell motility in a variety of recent studies, yet the role of its lipid product, phosphatidylinositol 1,4,5-trisphosphate (PtdIns-3,4,5-P3), has yet to be elucidated. In this study, three independent preparations of PtdIns-3,4,5-P3 were found to increase the motility of NIH 3T3 cells when examined utilizing a microchemotaxis chamber. Dipalmitoyl L-alpha-phosphatidyl-D-myo-inositol 3,4,5-triphosphate (Di-C16-PtdIns-3,4,5-P3) also produced actin reorganization and membrane ruffling. Cells pretreated with 12-O-tetradecanoylphorbol-13-acetate to cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility induced by Di-C16-PtdIns-3,4,5-P3. These results are consistent with previous observations that PtdIns-3,4,5-P3 activates Ca2+-independent PKC isoforms in vitro and in vivo and provide the first demonstration of an in vivo role for the lipid products of the phosphoinositide 3-kinase. PtdIns-3,4,5-P3 appears to directly initiate cellular motility via activation of a PKC family member.

3T3 Cells↗

PubMed1997

Characterization of factor-independent variants derived from TF-1 hematopoietic progenitor cells: the role of the Raf/MAP kinase pathway in the anti-apoptotic effect of GM-CSF.

Human hematopoietic progenitor cells (TF-1) undergo apoptosis upon deprivation of their dependent cytokine. In this report, we have isolated and characterized some spontaneously derived cytokine-independent variants from TF-1 cells. Analysis of several signaling molecules known to be activated by the GM-CSF pathway revealed that two non-autocrine variants were still responsive to GM-CSF stimulation. However, both variants, without ligand stimulation, already had some activated forms of Raf and MAP kinases. Given current knowledge, the activated Raf/MAP kinase pathway was likely to be responsible for the survival of both variants in the cytokine-free medium. However, the growth of hybrids between wild type and either variant was unexpectedly dependent on GM-CSF. Both variants like the wild type cells were still susceptible to apoptosis induced by other stimuli. These results suggest that either the activated Raf/MAP kinase pathway in both variants is not sufficient to repress the 'two-fold' death signals generated from the hybrids or that there is another mechanism that is responsible for the factor-independent growth of both variants.

Apoptosis↗

PubMed1997

Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure.

We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.

Acrylamide↗

PubMed1997

A continuous method for measuring calpain activity.

A simplified methodology for assaying the caseinolysis by calpains was developed. This methodology, including the incubation of calpain with casein and direct measurement of the absorbance at 500 nm, is based on the turbidity of the reaction mixture caused by the aggregation of hydrolysates during the reaction. Unlike the typical caseinolysis assay, this novel assay does not need to separate the substrate from hydrolysates and can be continuously monitored in visible wavelength range. The activity of calpain is expressed by the maximum reaction velocity (delta A500/min) at 25 degrees C).

Animals↗

PubMed1997

Selective recognition of phosphatidylinositol 3,4,5-trisphosphate by a synthetic peptide.

The present study takes a novel approach to explore the mode of action of phosphoinositide 3-kinase lipid products by identifying a synthetic peptide W-NG(28-43) (WAAKIQASFRGHMARKK) that displays discriminative affinity with phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). This PtdIns(3,4,5)P3-binding peptide was discovered by a gel filtration-based binding assay and exhibits a high degree of stereochemical selectivity in phosphoinositide recognition. It forms a 1:1 complex with PtdIns(3,4,5)P3 with Kd of 2 microM, but binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) with substantially lower affinity (5- and 40-fold, respectively) despite the largely shared structural motifs with PtdIns(3,4,5)P3. Other phospholipids examined, including phosphatidylserine, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, show low or negligible affinity with the peptide. Several lines of evidence indicate that this phosphoinositide-peptide interaction is not due to nonspecific electrostatic interactions or phospholipid aggregation, and requires a cooperative action among the hydrophobic and basic residues to exert the selective recognition. CD data suggest that the peptide acquires an ordered structure upon binding to PtdIns(3,4,5)P3. Further, we demonstrate that PtdIns(3,4,5)P3 enhances the phosphorylation rate of this binding peptide by protein kinase C (PKC)-alpha in a dose-dependent manner. In view of the findings that this stimulatory effect is not noted with other PKC peptide substrates lacking affinity with PtdIns(3,4,5)P3 and that PKC-alpha is not susceptible to PtdIns(3,4,5)P3 activation, the activity enhancement is thought to result from the substrate-concentrating effect of the D-3 phosphoinositide, i.e. the presence of PtdIns(3,4,5)P3 allows the peptide to bind to the same vesicles/micelles to which PKC is bound. Moreover, it is noteworthy that neurogranin, the full-length protein of W-NG(28-43) and a relevant PKC substrate in the forebrain, binds PtdIns(3,4,5)P3 with high affinity. Taken together, it is plausible that, in addition to PKC activation, PtdIns(3,4,5)P3 provides an alternative mechanism to regulate PKC activity in vivo by recruiting and concentrating its target proteins at the interface to facilitate the subsequent PKC phosphorylation.

Amino Acid Sequence↗

PubMed1997

Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells.

N-[5-(5, 7-dimethyl Bodipy)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C5-DMB-SM), a fluorescent analog of sphingomyelin, has been used in a study of the formation of very early endosomes in human skin fibroblasts. This lipid exhibits a shift in its fluorescence emission maximum from green (approximately 515 nm) to red (approximately 620 nm) wavelengths with increasing concentrations in membranes. When cells were incubated with 5 microM C5-DMB-SM at 4 degrees C and washed, only plasma membrane fluorescence (yellow-green) was observed. When these cells were briefly (< or = 1 min) warmed to 37 degrees C to allow internalization to occur, and then incubated with defatted bovine serum albumin (back-exchanged) at 11 degrees C to remove fluorescent lipids from the plasma membrane, C5-DMB-SM was distributed in a punctate pattern throughout the cytoplasm. Interestingly, within the same cell some endosomes exhibited green fluorescence, whereas others emitted red-orange fluorescence. Furthermore, the red-orange endosomes were usually seen at the periphery of the cell, while the green endosomes were more uniformly distributed throughout the cytoplasm. This mixed population of endosomes was seen after internalization times as short as 7 s and was also seen over a wide range of C5-DMB-SM concentrations (1-25 microM). Control experiments established that the variously colored endosomes were not induced by changes in pH, membrane potential, vesicle size, or temperature. Quantitative fluorescence microscopy demonstrated that the apparent concentration of the lipid analog in the red-orange endosomes was severalfold higher than its initial concentration at the plasma membrane, suggesting selective internalization (sorting) of the lipid into a subset of early endosomes. Colocalization studies using C5-DMB-SM and either anti-transferrin receptor antibodies or fluorescently labeled low-density lipoprotein further demonstrated that this subpopulation of endosomes resulted from receptor-mediated endocytosis. We conclude that the spectral properties of C5-DMB-SM can be used to distinguish unique populations of early endosomes from one another and to record dynamic changes in their number and distribution within living cells.

Cell Membrane↗