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Biomedical subjects

F Mao

Publications and source records attributed to F Mao.

At least 19 recordsLinked to original sources
PubMed2001

Tcf4 can specifically recognize beta-catenin using alternative conformations.

Accumulation of the Wnt pathway effector beta-catenin is a hallmark of a number of cancers, including colon cancer. As beta-catenin accumulates in the cell, it forms a complex with Tcf family transcription factors and activates the transcription of several critical genes involved in cell proliferation. Because Tcf4 is the predominant Tcf factor present in colon cancer cells, drugs that specifically disrupt the beta-catenin-Tcf4 complex could be useful in treating colon cancers. Earlier structural and biochemical studies demonstrated that the central region of the beta-catenin binding domain of Tcf is essential for anchoring Tcf to beta-catenin via two conserved lysines in beta-catenin (called the charged 'buttons'). Here we report the crystal structure of a beta-catenin-Tcf4 complex at 2.0 A resolution. Our structural and mutagenesis studies show that Tcf4 docks specifically to beta-catenin using several distinct conformations in its essential central region. These conformations allow different glutamate residues in the central region of Tcf4 to form a salt bridge with the same critical charged button, Lys 312 of beta-catenin. We propose that this interaction may be the first event in beta-catenin-Tcf4 recognition.

Amino Acid Sequence↗
PubMed2000

Crystal structure of a beta-catenin/Tcf complex.

The Wnt signaling pathway plays critical roles in embryonic development and tumorigenesis. Stimulation of the Wnt pathway results in the accumulation of a nuclear beta-catenin/Tcf complex, activating Wnt target genes. A crystal structure of beta-catenin bound to the beta-catenin binding domain of Tcf3 (Tcf3-CBD) has been determined. The Tcf3-CBD forms an elongated structure with three binding modules that runs antiparallel to beta-catenin along the positively charged groove formed by the armadillo repeats. Structure-based mutagenesis defines three sites in beta-catenin that are critical for binding the Tcf3-CBD and are differentially involved in binding APC, cadherin, and Axin. The structural and mutagenesis data reveal a potential target for molecular drug design studies.

Amino Acid Motifs↗
PubMed2000

Prostate boundary segmentation from 2D ultrasound images.

Outlining, or segmenting, the prostate is a very important task in the assignment of appropriate therapy and dose for cancer treatment; however, manual outlining is tedious and time-consuming. In this paper, an algorithm is described for semiautomatic segmentation of the prostate from 2D ultrasound images. The algorithm uses model-based initialization and the efficient discrete dynamic contour. Initialization requires the user to select only four points from which the outline of the prostate is estimated using cubic interpolation functions and shape information. The estimated contour is then deformed automatically to better fit the image. The algorithm can easily segment a wide range of prostate images, and contour editing tools are included to handle more difficult cases. The performance of the algorithm with a single user was compared to manual outlining by a single expert observer. The average distance between semiautomatically and manually outlined boundaries was found to be less than 5 pixels (0.63 mm), and the accuracy and sensitivity to area measurements were both over 90%.

Algorithms↗
PubMed2000

Segmentation of carotid artery in ultrasound images: method development and evaluation technique.

Segmentation of carotid artery lumen in two-dimensional and three-dimensional ultrasonography is an important step in computerized evaluation of arterial disease severity and in finding vulnerable atherosclerotic plaques susceptible to rupture causing stroke. Because of the complexity of anatomical structures, noise as well as the requirement of accurate segmentation, interactions are necessary between observers and the computer segmentation process. In this paper a segmentation process is described based on the deformable model method with only one seed point to guide the initialization of the deformable model for each lumen cross section. With one seed, the initial contour of the deformable model is generated using the entropy map of the original image and mathematical morphology operations. The deformable model is driven to fit the lumen contour by an internal force and an external force that are calculated, respectively, with geometrical properties of deformed contour and with the image gray level features. The evaluation methodology using distance-based and area-based metrics is introduced in this paper. A contour probability distribution (CPD) method for calculating distance-based metrics is introduced. The CPD is obtained by generating contours of the lumen using a set of possible seed locations. The mean contour can be compared to a manual outlined contour to provide accuracy metrics. The variance computed from the CPD can provide metrics of local and global variability. These metrics provide a complete performance evaluation of an interactive segmentation algorithm and a means for comparing different algorithm settings.

Algorithms↗
PubMed1999

Localization of carbonic anhydrase in living osteoclasts with bodipy 558/568-modified acetazolamide, a thiadiazole carbonic anhydrase inhibitor.

We describe the synthesis of Bodipy 558/568-modified acetazolamide, a fluorescent inhibitor of carbonic anhydrase and its use to localize the enzyme in living cells. The modified acetazolamide, with its specific sulfonamide group intact, labeled cells at concentrations as low as 10(-9) M, with a minimal loading time of 5 min. The staining was decreased by 57.4% by preincubating cells with unaltered acetazolamide (1:100) or with trifluoromethane sulfonamide, 6-ethoxyzolamide, and 5-(3-hydroxybenzoyl)-thiophene-2-sulfonamide. The efficacy of the inhibitor was unchanged by the fluorescent label, as determined by an acridine orange assay that detects acidification of osteoclasts, the cell model used in this study. This compound should prove to be useful for studying carbonic anhydrase in many organisms because of the high degree of conservation of the active site of this enzyme. (J Histochem Cytochem 47:545-550, 1999)

Acetazolamide↗
PubMed1999

Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.

Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the Alexa names indicate the approximate excitation wavelength maximum in nm). All Alexa dyes and their conjugates are more fluorescent and more photostable than their commonly used spectral analogues listed above. In addition, Alexa dyes are insensitive to pH in the 4-10 range. We evaluated Alexa dyes compared with conventional dyes in applications using various conjugates, including those of goat anti-mouse IgG (GAM), streptavidin, wheat germ agglutinin (WGA), and concanavalin A (ConA). Conjugates of Alexa 546 are at least twofold more fluorescent than Cy3 conjugates. Proteins labeled with the Alexa 568 or Alexa 594 dyes are several-fold brighter than the same proteins labeled with lissamine rhodamine B or Texas Red dyes, respectively. Alexa dye derivatives of phalloidin stain F-actin with high specificity. Hydrazide forms of the Alexa dyes are very bright, formaldehyde-fixable polar tracers. Conjugates of the Alexa 430 (ex 430 nm/em 520 nm) and Alexa 532 (ex 530 nm/em 548 nm) fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges.

Animals↗
PubMed1998

Fragmentary window filtering for multiscale lung nodule detection: preliminary study.

RATIONALE AND OBJECTIVES: The authors evaluated computer-assisted diagnostic (CAD) methods used to detect suspicious areas on lung radiographs. MATERIALS AND METHODS: The authors designed a fragmentary window filtering (FWF) algorithm for detecting lung nodule patterns, which generally appear as circular areas of high opacity on the chest radiograph. The FWF algorithm helps differentiate circular patterns from overlapping radiographic background. A multiscale analysis was performed to locate multiscale nodules. Receiver operating characteristic analysis was performed by using a lung nodule that was extracted from a chest radiograph. The nodule underwent scalings and subsequent superimposition onto 140 normal regions of interest from six chest radiographs. RESULTS: The FWF method was superior to the matched filtering method in the detection of suspicious areas. CONCLUSION: The proposed FWF-based method should provide improved detection of lung nodules on chest radiographs.

Algorithms↗
PubMed1997

Developmental expression and regulation of alpha 1 adrenergic receptors in cultured cortical neurons.

The expression and distribution of alpha 1 receptors in cultured neurons derived from rat visual cortex were studied with confocal microscopy using the fluorescently labeled alpha 1 adrenergic receptor selective antagonist BODIPY FL prazosin. The receptors were found to be clustered on neuronal somata and on proximal dendrites. We found that expression of the alpha 1 receptor is regulated both by neuronal excitability and by the usage of the receptor itself. The specificity of receptor regulation to the blockade of a particular receptor class was also studied. We approached this by comparing the effects of treatment with the alpha 1 adrenoreceptor antagonist prazosin and M1 muscarinic receptor antagonist pirenzepine on the expression of alpha 1 and M1 receptors in cultured cortical neurons. The results showed that blockade of muscarinic receptors with pirenzepine up-regulated muscarinic receptor expression selectively without changing alpha 1 receptor expression. Conversely blockade of alpha 1 receptors up-regulated alpha 1 expression but not muscarinic receptor expression. This implies that the expression levels of M1 and alpha 1 receptors are both regulated through specific signal transduction pathways. The interactions between neuronal activity and receptor activation (or blockade) on receptor expression were studied as well. In these experiments we compared the effects of high K+, tetrodotoxin (TTX), prazosin, and noradrenaline on the expression of alpha 1 and M1 receptors in cultured neurons. The results show that high K+ exposure increased both alpha 1 and M1 receptor expression regardless of the presence of receptor agonists or antagonists. On the other hand, TTX exposure reduced both alpha 1 and M1 receptor expression regardless of the presence of the receptor agonists or antagonists. This implies that regulation of receptor number is predominantly regulated by neuronal activity rather than by receptor occupancy.

Animals↗
PubMed1997

Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes.

BACKGROUND & AIMS: Bile salt-induced apoptosis is mediated by a trypsin-like nuclear protease. The aims of this study were to identify this protease and to elucidate its mechanistic role in bile salt-induced hepatocyte apoptosis. METHODS: Rats, isolated rat hepatocytes, and a rat hepatoma cell line stably transfected with a bile salt transporter (McNtcp.24) were used for this study. RESULTS: In the bile duct-ligated rat, a threefold increase in apoptosis and a fourfold increase in trypsin-like nuclear protease activity were observed. The nuclear protease activity was purified from bile duct-ligated rats and identified as cathepsin B. Specific, structurally dissimilar cathepsin B inhibitors blocked glycochenodeoxycholate (GCDC)-induced apoptosis in cultured rat hepatocytes. Furthermore, stable transfection of McNtcp.24 cells with the complementary DNA for cathepsin B in the antisense orientation reduced cathepsin B activity and GCDC-induced apoptosis by >75%. Next, cathepsin B cellular localization during apoptosis was determined by immunoblot analysis of nuclear cell fractions, immunocytochemistry, and by determining the compartmentation of expressed cathepsin B fused to green fluorescent protein. All three approaches showed translocation of cathepsin B from the cytoplasm to the nucleus during GCDC-induced apoptosis. CONCLUSIONS: The data suggest that translocation of cathepsin B from the cytoplasm to the nucleus is a mechanism contributing to bile salt-induced apoptosis of hepatocytes.

Animals↗
PubMed1997

Cholesterol deposition in macrophages: foam cell formation mediated by cholesterol-enriched oxidized low density lipoprotein.

Oxidized low density lipoprotein (LDL) is thought to mediate the transformation of macrophages to cholesterol-rich foam cells. Yet convincing evidence for this process is lacking in vitro. We suggest that oxidized LDL-mediated foam cell formation is not seen in vitro because the cholesteryl ester content of LDL particles (oxidized in the presence of transition metals) is dramatically reduced. Thus, if oxidized LDL could be cholesterol-enriched prior to its addition to macrophages, this lipoprotein would be made more capable of inducing the cellular deposition of cholesteryl esters. When we enriched cupric sulfate-oxidized LDL with cholesterol by incubation of this lipoprotein with unesterified cholesterol/phosphatidylcholine liposomes and added it to mouse peritoneal macrophage cultures, we found that: a) the enrichment of oxidized LDL with cholesterol did not alter the extent of oxidized LDL degradation; b) the cells accumulated massive amounts of cholesteryl ester (148 microg/mg cell protein) and unesterified cholesterol (260 microg/mg cell protein) after 24 h of incubation; and c) Sephacryl S-1000 chromatography of the cholesterol-enriched oxidized LDL verified the formation of large oxidized LDL-unesterified cholesterol/phosphatidylcholine complexes. These results demonstrate that oxidized LDL, when cholesterol-enriched, can mediate the formation of macrophage foam cells in culture

Animals↗
PubMed1997

Spatio-temporal grouping for the formation of vascular segments in coronarography image sequences.

This work is aimed at the extraction of vascular structures using perceptual grouping methods. The approach is based on structural feature matching, between images in the time sequence. These features correspond to junctions (or bifurcation points) and are paired by means of a local criteria. A global refinement procedure is then applied by means of a relaxation scheme. The feature trajectories over the entire image sequence are examined to further build sets of vascular segments, which better describe vascular branches. The resulting performances are exemplified on a standard biplane coronarographic examination.

Angiography, Digital Subtraction↗
PubMed1996

Selective induction of apoptosis in Hep 3B cells by topoisomerase I inhibitors: evidence for a protease-dependent pathway that does not activate cysteine protease P32.

Progress in the treatment of hepatocellular carcinoma (HCC), a common tumor worldwide, has been disappointing. Inhibitors of topoisomerases are being widely studied as potential inducers of tumor cell apoptosis. Our aims were to determine whether topoisomerase-directed drugs would induce apoptosis in a human HCC cell line (Hep 3B) and, if so, to investigate the mechanism. The topoisomerase I poison camptothecin (CPT) induced apoptosis of Hep 3B cells in a time- and concentration-dependent manner. In contrast, the topoisomerase II poison etoposide failed to induce apoptosis despite the apparent stabilization of topoisomerase II-DNA complexes. Unexpectedly, CPT-induced apoptosis in this cell type occurred without any detectable cleavage of poly(ADP-ribose) polymerase or lamin B, polypeptides that are commonly cleaved in other cell types undergoing apoptosis. Likewise, Hep 3B cell apoptosis occurred without a detectable increase in interleukin-1beta-converting enzyme (ICE)-like or cysteine protease P32 (CPP32)-like protease activity. In contrast, trypsin-like protease activity (cleavage of Boc-Val-Leu-Lys-chloromethylaminocoumarin in situ) increased threefold in cells treated with CPT but not etoposide. Tosyl-lysyl chloromethyl ketone inhibited the trypsin-like protease activity and diminished CPT-induced apoptosis. These data demonstrate that (a) apoptosis is induced in Hep 3B cells after stabilization of topoisomerase I-DNA complexes but not after stabilization of topoisomerase II-DNA complexes as measured by alkaline filter elution; (b) Hep 3B cell apoptosis occurs without activation of ICE-like and CPP32-like protease activity; and (c) a trypsin-like protease activity appears to contribute to apoptosis in this cell type.

Apoptosis↗
PubMed1996

Iron-ascorbate-phospholipid mediated modification of low density lipoprotein.

LDL can be oxidized by a variety of agents to form a modified lipoprotein which is capable of being avidly metabolized by macrophages. While previous in vitro studies have focused exclusively on the oxidation of LDL, other lipids found in the atheroma are also subject to oxidation and its lipoperoxide byproducts may contribute to the process of LDL modification. To examine the relationship between the oxidation of phospholipids and the subsequent modification of LDL, we incubated 250 microM phosphatidylcholine with 10 microM ferrous sulfate and 50 microM ascorbic acid in 10 mM Tris (pH 7.0). After 18 h at 37 degrees C, significant amounts of thiobarbituric acid reactive substances (TBARS) were formed. The inclusion of LDL (100 micrograms protein/ml) elevated the TBARS and increased the electrophoretic mobility of the lipoprotein. LDL treated with iron and ascorbate in the absence of phosphatidylcholine did not result in the modification of this lipoprotein. LDL that was incubated with phosphatidylcholine, iron and ascorbate was found to be metabolized by macrophages to a far greater extent than native LDL or LDL treated with phosphatidylcholine alone. Probucol (10 microM) inhibited the LDL modification process. These results demonstrate that while iron and ascorbate cannot oxidize LDL directly, the addition of phosphatidylcholine to these initiators of lipid peroxidation can mediate and lead to the modification of LDL.

Animals↗
PubMed1996

Characterization of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes.

A novel membrane aminopeptidase has been identified as a major protein in vesicles from rat adipocytes containing the glucose transporter isotype Glut4. In this study we have characterized this aminopeptidase, referred to as vp165, in 3T3-L1 adipocytes. The subcellular distributions of vp165 and Glut4 were determined by immunoisolation of vesicles with antibodies against both proteins, by immunofluorescence, and by subcellular fractionation and immunoblotting. Relative amounts of vp165 at the cell surface in basal and insulin-treated cells were assayed by cell surface biotinylation. These experiments showed that vp165 and Glut4 were entirely colocalized and that vp165 increased markedly at the cell surface in response to insulin, in a way similar to Glut4. When intact cells were assayed with a novel, membrane-impermeant fluorogenic substrate for vp165, we found that insulin stimulated aminopeptidase activity at the cell surface. This observation provides direct evidence for the functional consequence of vp165 translocation.

3T3 Cells↗
PubMed1996

Imaging exocytosis and endocytosis.

From the secretion of neurotransmitters via synaptic vesicles to the expulsion of cellular waste via contractile vacuoles, exocytosis and its sequel, endocytosis, are being explored with a variety of new optical tools. Fluorescent markers, especially styryl dyes such as FM1-43 (which reversibly labels endosomal membranes), have been used to follow exo- and endocytic events in many cell types. Even though the development of new dyes is still largely empirical, some theoretical principles have emerged to guide future dye chemistry. Moreover, advances in optical imaging technology that augment conventional fluorescence microscopy are appearing. For example, interference reflection microscopy (which requires no flurophore) and total internal reflection microscopy have recently been used to observe single exocytic events at the contact point between a glass coverslip and the plasma membrane.

Animals↗
PubMed1995

Association of negatively-charged phospholipids with low-density lipoprotein (LDL) increases its uptake and the deposition of cholesteryl esters by macrophages.

LDL, the major carrier of cholesterol in blood, is poorly metabolized by macrophages. In contrast, macrophages can recognize and endocytose anionic phospholipids such as phosphatidylserine, phosphatidylglycerol and cardiolipin. Since macrophages can take up large amounts of these phospholipids, experiments were performed to ascertain whether pre-incubation of native LDL with negatively-charged phospholipids would enhance the metabolism of LDL by macrophages. When 125I-LDL was incubated with cardiolipin liposomes for 18 h at 37 degrees C before addition to macrophages, an approx. 40-fold increase of LDL metabolism by these cells was observed. Similar results were found when LDL was pre-incubated with phosphatidylserine or phosphatidylglycerol; however, pre-incubation of LDL with phosphatidylcholine liposomes did not lead to an increase of LDL metabolism. The macrophage uptake of LDL pre-incubated with cardiolipin was reduced to approx. 40% of control values in the presence of dextran sulfate and fucoidin, inhibitors of anionic phospholipid uptake. Cytochalasin D, an inhibitor of phagocytosis, reduced the lysosomal degradation of LDL pre-incubated with cardiolipin to approx. 10% of control values. When the LDL-cardiolipin mixture was chromatographed on agarose gel, two peaks containing LDL were observed in the elution profile: the first peak appeared at the void volume and the second peak was detected just ahead of native LDL. The LDL in both peaks was much more extensively metabolized by macrophages than was native LDL; the LDL in the first peak was metabolized at a rate that was 8 times the second peak. The results demonstrate that negatively-charged phospholipids can form a complex with LDL which facilitates its phagocytosis by macrophages.

Cardiolipins↗
PubMed1995

Synthesis of a photoaffinity analog of 3'-azidothymidine, 5-azido-3'-azido-2',3'-dideoxyuridine. Interactions with herpesvirus thymidine kinase and cellular enzymes.

Long term administration of 3'-azidothymidine (AZT) for the treatment of AIDS has led to detrimental clinical side effects in some patients, the biochemical causes of which are still being delineated. Base-substituted, azido-nucleotide photoaffinity analogs have routinely proven to be effective tools for identifying and characterizing nucleotide-utilizing enzymes. Therefore, we have synthesized 5-azido-3'-azido-2',3'-dideoxyuridine, which is a potential photoaffinity analog of two human immunodeficiency virus drugs, AZT and 3'azido-2',3'-dideoxyuridine. A partially purified herpes simplex virus type 1 thymidine kinase and [gamma-32P]ATP were used to make an AZT monophosphate analog, [32P]5-azido-3'-azido-2',3'-dideoxyuridine monophosphate. The photoaffinity properties of this analog were initially tested with herpes simplex virus type 1 thymidine kinase. Photoaffinity labeling of this enzyme was saturable (half-maximal, 30 microM) and could be specifically inhibited by AZT, AZT monophosphate, thymidine, and thymidine monophosphate. Photolabeling of rat liver microsomal membranes was also done, and several membrane proteins that interact with AZT monophosphate were identified. The antiviral and cytotoxic activities of 5-azido-3'-azido-2',3'-dideoxyuridine were determined using human immunodeficiency virus, type 1 strain IIIB and an AZT drug-resistant strain in human T lymphocyte H9 cells.

Affinity Labels↗