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Y Yeh

Publications and source records attributed to Y Yeh.

At least 37 records · Page 2Linked to original sources
PubMed1994

Membrane proteases as potential diagnostic and therapeutic targets for breast malignancy.

Metastasizing cancer cells can invade the extracellular matrix using plasma membrane protrusions, termed invadopodia, that contact and dissolve the matrix. Various membrane associated proteases localized on the invadopodial membranes are responsible for the extracellular matrix degradation. Work from our laboratory shows that secreted proteases including Gelatinase A, and high molecular weight integral membrane proteases are associated with cell surface invadopodia. Three cell types, including chicken embryonic cells transformed by Rous sarcoma virus, human malignant melanoma cell line LOX, and human breast carcinoma cell line MDA-MB-231, retain the invasive phenotype in vitro, express invadopodia, degrade and enter into a fibronectin-rich collagenous matrix. We suggest that invadopodium-associated proteases are ideal targets for the diagnosis and treatment of cancer as their presence in association with primary tumors may signal increased metastatic potential. An approach toward the development of new prognostic markers for breast malignancy involved production of monoclonal antibodies directed against membrane proteases in a mixture of glycoproteins. Double immunofluorescent technique using a known invadopodium marker is designed to select specific monoclonal antibodies colocalizing at the invasion front, on invadopodia of cancer cells. Membrane protease accessibility at the cell surface can therefore be exploited for therapeutic advances by the development of specific antibodies and inhibitors that block their activities, and by the use of monoclonal antibodies to target cytotoxic molecules to micrometastases. Also, this same accessibility may potentially be used to detect surface proteases on micrometastases or to detect components shed by micrometastases in serum.

Antigens, Neoplasm↗
PubMed1994

Rigorous analysis of light diffraction ellipsometry by striated muscle fibers.

A rigorous analysis of both the transverse electric and the transverse magnetic modes of light diffracted from a muscle fiber is performed. From the expressions of electromagnetic field components, ellipsometry parameters, differential field ratio, r, and birefringence, delta n, have been obtained. A theoretical formulation that introduces myofibril skew planes and a randomization factor about the average skew plane yields a relationship that shows good fit to experimental data of Chen et al. (Biophys. J. 56:595, 1989) and Burton et al. (J. Muscle Res. Cell Motil. 11:258, 1990). Using indices of refraction within each of the regions of the sarcomeric unit that are consistent with our knowledge of the molecular structure of the sarcomere in the analysis, it is shown that the transition from the rigor state to the resting state leads to as much as a approximately 13% decrease in the r-value and an equally significant change in delta n.

Animals↗
PubMed1994

Elevation of glutathione levels by phase II enzyme inducers: lack of inhibition of human immunodeficiency virus type 1 replication in chronically infected monocytoid cells.

Supplementation of media with high concentrations of thiols (5-20 mM) inhibits human immunodeficiency virus type 1 (HIV-1) replication in vitro. Compounds that prevent carcinogenesis via induction of phase II enzymes also elevate intracellular GSH levels, thus raising the possibility that chemopreventive enzyme inducers may represent a more pharmacologically feasible method to inhibit viral replication. Previous studies revealed that oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] was the only GSH inducer tested that could inhibit HIV-1 replication in acutely infected H9 cells. Because thiols are proposed to suppress transcription of the integrated HIV-1 genome by preventing the activation of nuclear factor-kappa B, experiments evaluating inducers of GSH levels in acutely infected H9 cells do not rule out the ability of these compounds to inhibit viral replication in chronically infected cells exposed to cytokines or mitogens. Therefore, we determined the antiviral effects of several inducers in phorbol-12-myristate-13- acetate-stimulated U1 cells, a monocytoid cell line that contains two integrated copies of the HIV-1 genome. Although 1,2-dithiole-3-thione, dimethyl fumarate, and oltipraz can elevate cytosolic thiol levels, only oltipraz inhibited HIV-1 replication. Moreover, decreased nuclear factor-kappa B binding activity could be correlated with increases in cytosolic thiols produced by various treatments (r2 = 0.8) but not with suppression of viral replication (r2 = 0.01). These data suggest that oltipraz-induced increases in GSH are not responsible for the antiviral action of oltipraz and that elevation of intracellular GSH levels by chemopreventive enzyme inducers does not inhibit viral replication.

Antiviral Agents↗
PubMed1993

Force-velocity relationships in kinesin-driven motility.

Kinesin is a microtubule-based motor protein that uses energy released from Mg-ATP hydrolysis to generate force for the movement of intracellular membranes towards the fast-growing (plus) ends of microtubule tracks in cells. Kinesin-driven microtubule movement can be visualized and quantified using light microscope motility assays but our understanding of how kinesin generates force and motion is incomplete. Here we report the use of a centrifuge microscope to obtain force-velocity curves for kinesin-driven motility and to estimate that the maximal isometric force generated per kinesin is 0.12 +/- 0.03 pN per molecule.

Adenosine Triphosphate↗
PubMed1993

Binding and localization of M(r) 72,000 matrix metalloproteinase at cell surface invadopodia.

Degradation (turnover) of collagenous matrix occurs on the surface of specialized membrane extensions termed "invadopodia," which are sites of cell invasion into the extracellular matrix. Here we show the localization of the M(r) 72,000 type IV collagenase of the matrix metalloproteinase family at invadopodia. When added exogenously, latent M(r) 72,000 collagenase binds to invadopodia of chicken embryo fibroblasts transformed by Rous sarcoma virus, whereupon the bound collagenase loses its propeptide. The collagenase binds to a component contained within the detergent extract of transformed cells, and increased levels of the active M(r) 62,000 form of the collagenase are seen here. Such an association is not detected in the detergent extract derived from normal cells. Using a recently developed cell fractionation procedure to collect cell surfaces enriched in invadopodia, we show that the M(r) 72,000 collagenase associates with the invadopodial fraction and active forms of the enzyme become immobilized on the collagenous surface. Thus, invadopodia direct intense localized degradation of the extracellular matrix by concentrating active membrane-associated collagenases at sites of cellular invasion.

Amino Acid Sequence↗
PubMed1993

Oltipraz, an inhibitor of human immunodeficiency virus type 1 replication.

Glutathione depletion may play a pivotal role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1) infection. Since certain compounds prevent experimental carcinogenesis by elevating the levels of glutathione and phase II detoxication enzymes, we compared the potencies of several inducers with their ability to inhibit basal levels of HIV-1 replication in H9 cutaneous T-cell lymphoma cells. All monofunctional inducers tested elevated the levels of glutathione and quinone reductase, a marker for phase II enzyme induction. However, only oltipraz [4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione] was effective at inhibiting HIV-1 replication (IC50 = 14.8 +/- 3.1 microM). The antiviral effect of oltipraz was potentiated by 3'-azido-3'-deoxythymidine. Thus, 1,2-dithiole-3-thiones represent a hitherto unrecognized class of anti-HIV-1 agents. Oltipraz behaves kinetically as an irreversible inhibitor of HIV-1 reverse transcriptase in the template-primer binding domain. Oltipraz has been used to treat schistosomiasis in humans and is undergoing clinical evaluation as an anticarcinogen. Thus, oltipraz (and other 1,2-dithiole-3-thiones) may have therapeutic utility in HIV-1-infected individuals, not only because of their antiretroviral activity, but also by preventing the development of HIV-1-associated neoplasms.

Antiviral Agents↗
PubMed1993

Characteristics, substrate analysis, and intracellular location of Saccharomyces cerevisiae UMP kinase.

The yeast Saccharomyces cerevisiae SOC8 DNA fragment was isolated as a wildtype dominant suppressor of the cdc8 mutation. The SOC8 has previously been proved to be allelic with URA6, encoding the UMP kinase in yeast cells. The protein has been purified to homogeneity. In this report, we describe the characteristics of the UMP kinase from yeast. The yeast enzyme requires a divalent cation and is active over the entire range of pH from 6 to 9.5. The enzyme can use UMP and dUMP as phosphate acceptors with high activity; to a lesser extent, it can also use IMP, GMP, dGMP, 5-iodo-dUMP, XMP, and dTMP as substrates. ATP and dATP are the best phosphate donors; the enzyme could use GTP, dGTP, dCTP, and dTTP to some degree (30-50%). CTP and UTP were poor phosphate donors for the UMP kinase reaction (10-14%). Like other monophosphate kinases, UMP kinase contains a conserved nucleotide-binding site at its N-terminus following a cysteine residue, and its enzymatic activity is inhibited by sulfhydryl inhibitors such as 5,5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide. Subcellular localization studies indicate that the UMP kinase locates primarily in the cytoplasm (approximately 80%) and also in the nucleus (approximately 20%), but not in the mitochondria. These results suggest that it may exert its function in the nucleus, such as in RNA synthesis, as well as in the cytoplasm, but not in the mitochondria. The presence of UMP kinase in the nucleus might facilitate its suppression of cdc8 mutant cells, which are defective in nuclear DNA synthesis.

Amino Acid Sequence↗
PubMed1992

Rigorous analysis of light diffraction by a striated muscle fibre.

A rigorous theory describing the diffraction of light by a muscle fibre has been formulated. The basis of this analysis is the rigorous coupled-wave approach of T. K. Gaylord & M. G. Moharam (Proc. IEEE 73, 894 (1985)); however, we obtain here a closed-form analytical result that is both mathematically simple and physically easy to understand. We have compared our results on striated muscle fibres with the analytical results obtained by A. F. Huxley (Proc. R. Soc. Lond. B 241, 65 (1990)) using the normal mode approach, and with those obtained by R. A. Thornhill, N. Thomas & N. Berovic (Eur. Biophys. J. 20, 87 (1991)) using a multiwave first-order coupled-wave approximation. For an equivalent set of assigned fibre parameters, our results are consistent with these mentioned. Extension of this analysis to a fibre with different structures showed that the differences in diffraction efficiencies of different orders for a frog skeletal fibre and for an insect flight fibre are clear; the sensitivity to distinct structural organization of the fibre is very good.

Algorithms↗
PubMed1992

Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells.

Tyrosine phosphorylation of membrane-associated proteins is involved at two distinct sites of contact between cells and the extracellular matrix: adhesion plaques (cell adhesion and de-adhesion) and invadopodia (invasion into the extracellular matrix). Adhesion plaques from chicken embryonic fibroblasts or from cells transformed by Rous sarcoma virus contain low levels of tyrosine-phosphorylated proteins (YPPs) which were below the level of detection in 0.5-microns thin, frozen sections. In contrast, intense localization of YPPs was observed at invadopodia of transformed cells at sites of degradation and invasion into the fibronectin-coated gelatin substratum, but not in membrane extensions free of contact with the extracellular matrix. Local extracellular matrix degradation and formation of invadopodia were blocked by genistein, an inhibitor of tyrosine-specific kinases, but cells remained attached to the substratum and retained their free-membrane extensions. Invadopodia reduced or lost YPP labeling after treatment of the cells with genistein, but adhesion plaques retained YPP labeling. The plasma membrane contact fractions of normal and transformed cells have been isolated form cells grown on gelatin cross-linked substratum using a novel fractionation scheme, and analyzed by immunoblotting. Four major YPPs (150, 130, 81, and 77 kD) characterize invadopodial membranes in contact with the matrix, and are probably responsible for the intense YPP labeling associated with invadopodia extending into sites of matrix degradation. YPP150 may be an invadopodal-specific YPP since it is approximately 3.6-fold enriched in the invasive contact fraction relative to the cell body fraction and is not observed in normal contacts. YPP130 is enriched in transformed cell contacts but may also be present in normal contacts. The two major YPPs of normal contacts (130 and 71 kD) are much lower in abundance than the major tyrosine-phosphorylated bands associated with invadopodial membranes, and likely represent major adhesion plaque YPPs. YPP150, paxillin, and tensin appear to be enriched in the cell contact fractions containing adhesion plaques and invadopodia relative to the cell body fraction, but are also present in the soluble supernate fraction. However, vinculin, talin, and alpha-actinin that are localized at invadopodia, are equally concentrated in cell bodies and cell contacts as is the membrane-adhesion receptor beta 1 integrin. Thus, tyrosine phosphorylation of the membrane-bound proteins may contribute to the cytoskeletal and plasma membrane events leading to the formation and function of invadopodia that contact and proteolytically degrade the extracellular matrix; we have identified several candidate YPPs that may participate in the regulation of these processes.

Actins↗
PubMed1991

Effect of boronic acids on antifreeze proteins.

The activity of antifreeze glycoprotein from the blood serum of Boreagadus saida was strongly inhibited by ions of organic boronic acids as well as by borate. The activity of nonglycoprotein from the blood serum of Pseudopleuronectus americanus, however, was not similarly inhibited. The inhibition by borate is thus specific for molecules with the carbohydrate moiety.

Animals↗
PubMed1991

The molecular origin of birefringence in skeletal muscle. Contribution of myosin subfragment S-1.

The state of optical polarization of He-Ne laser light diffracted by single skinned frog skeletal muscle fibers has been determined after decoration of the thin filaments of rigor fibers with exogenous S-1. Light on the first diffraction order was analyzed using optical ellipsometry for changes occurring in total birefringence (delta nT) and total differential field ratio (rT) and the experimental results compared with theoretical predictions. Fibers were examined with SDS-gel electrophoresis and electron microscopy as independent assays of S-1 binding. The binding of S-1 to the thin filaments caused a significant increase in rT and a small but significant decrease in delta nT. Release of bound exogenous S-1 with magnesium pyrophosphate demonstrated that the effect of S-1 on the optical parameters was reversible and both electrophoresis and electron microscopy demonstrated the presence of S-1 specifically bound to the thin filaments. Model simulations based on the theory of Yeh, Y., and R. Baskin (1988. Biophys. J. 54:205-218) showed that the values of delta nT and rT were sensitive to the axial bonding angle of exogenous S-1 as well as to the volume fraction of added S-1. Analysis of the data in light of the model showed that an average axial S-1 binding angle of 68 degrees +/- 7 degrees best fit the data.

Animals↗
PubMed1991

Lectin histochemistry of cystic jaw lesions: an aid for differential diagnosis between cystic ameloblastoma and odontogenic cysts.

The binding sites for Ulex europaeus agglutinin I (UEA-I), Bandeirea simplicifolia agglutinin I (BSA-I), and peanut agglutinin (PNA) were comparatively examined in the surgical materials from 41 cases of cystic and solid ameloblastomas and 42 cases of non-neoplastic odontogenic cysts including dentigerous cyst, odontogenic keratocyst, and radicular cyst. In non-neoplastic cysts, most of epithelial lining layers gave positive binding with UEA-I and BSA-I. However, no positive reactions were obtained for these two lectins in the epithelial components of ameloblastoma, except for limited UEA-I binding to markedly keratinized tumor cells in four cases. PNA binding was irregular and did not make any clear distinction between ameloblastomas and cysts. The results suggest that the lectin staining for UEA-I and BSA-I is a useful histologic aid for differential diagnosis between cystic ameloblastoma and non-neoplastic jaw cysts.

Ameloblastoma↗
PubMed1990

Diffraction ellipsometry studies of osmotically compressed muscle fibers.

Microstructural features of relaxed, skinned muscle fibers compressed with polyvinylpyrrolidone were examined by optical diffraction ellipsometry. This technique is sensitive to the optical anisotropy within the muscle, including that due to intrinsic properties of the protein molecules as well as that due to the regular arrangement of proteins in the surrounding medium. The change in polarization state of light after interacting with the muscle is described by the differential field ratio (DFR) and birefringence (delta n). Compression of single fibers (sarcomere length = 2.6 microns) with 0%-21% polyvinylpyrrolidone caused an increase of up to 23% and 31% for DFR and delta n, respectively. The largest increase in both parameters occurred at intermediate sarcomere lengths. Theoretical modelling of the results suggest that the average S-1 tilt angle may be reduced upon compression of the filament lattice. This is supported by experiments in which S-1 was enzymatically cleaved with alpha-chymotrypsin. Separate experiments comparing fibers with intact membranes and skinned fibers compressed to an equivalent lattice spacing showed little difference in DFR or delta n.

Animals↗
PubMed1990

Photon correlation spectroscopy of the polarization signal from single muscle fibres.

Measurements of crossbridge dynamics have been obtained from the spectrum of diffracted light derived from single, skinned fibres of skeletal muscle. This technique combines optical ellipsometry with photon correlation spectroscopy of the intrinsic reporters from the muscle cell. The difference in the intensities of the two linearly polarized electric field components of diffracted light is autocorrelated in time, and the dynamics of optical anisotropy from the contributing units of the sarcomere are measured. In focusing on the fast-time dynamics, we detected two principal features: a rapidly relaxing temporal signal with a relaxation time of approximately 5 microseconds, and an oscillatory component which transforms into a broad but spectrally defined signal centred at around 370 kHz. Experiments were conducted to measure changes of these two signals upon relax-rigor transition and alpha-chymotrypsin degradation. These studies strongly suggest that the relaxational component of the signal is primarily due to myosin crossbridge motion; the 370 kHz spectral signal has as its likely source the myosin thick filament.

Animals↗
PubMed1990

Crossbridge activity monitored from the state of polarization of light diffracted by activated frog muscle fibres.

The polarization properties of the first diffraction order have been measured when single frog fibres are illuminated by laser light. The relative difference in the amplitudes of the orthogonal electric field polarization components (differential field ratio) as well as their phase shift normalized by the pathlength (birefringence) have been obtained from fibres at rest and during fixed-end twitches and tetani. The differential field ratio decreased during contraction and the change during a single twitch averaged 69% of that during a companion tetanus. The birefringence of the first order averaged 2.80 +/- 0.59 x 10(-3) (mean +/- SD) at rest and the average decrease during a tetanus was 8.4% +/- 6.4%. The decrease in the differential field ratio upon activation was a decreasing function of sarcomere length, maximum at rest length and falling to zero at about 3.7 microns. Differences between the two first diffraction orders were observed for both the differentiated field ratio and the birefringence. At the time when force had risen to half the value reached at the end of the fast rise of tension, the change in the differential field ratio lead the tension by about 10-15 ms. The differential field ratio returned to its resting value after the fall of tension. The above results suggest that the differential field ratio is a sensitive indicator of intact fibre structure. The temporal lead in the differential field ratio with respect to tension rise supports models in which crossbridges initially attach in a non-force-producing state.

Animals↗
PubMed1989

Polarization states of diffracted light. Changes accompanying fiber activation.

Measurement of the state of optical polarization of light diffracted from single, skinned and intact fibers of anterior tibialis muscle from Rana pipiens revealed a dependence upon rigor, activation, and sarcomere length (SL) change. Changes in total birefringence, delta nT, and differential field ratio value, rT, were determined. In a relaxed, skinned fiber the total birefringence value, delta nT, decreases as sarcomere length is increased from 2.1 microns to approximately 2.8-3.0 microns. From there it increases significantly to a value of approximately 1.8 x 10(-3) at a sarcomere length of 3.6 microns. The differential field ratio, rT, also shows a biphasic response to increasing sarcomere length, first exhibiting a rapid decrease over shorter SL and leveling out after the SL is beyond 3.0 microns. In comparison, relaxed intact fibers change substantially less upon sarcomere length change, showing little change in birefringence and a small bi-phasic change in rT. Skinned fibers were activated using a solution that has the same ionic strength as the relaxing solution and allows repeatable, and sustained activation. A decrease in both delta nT and rT was observed upon fiber activation. The decrease in delta nT and rT was slightly larger at shorter sarcomere lengths than at longer lengths. Relaxed fibers placed in rigor showed changes in delta nT and rT similar to those observed in activated fibers. These results are consistent with the hypothesis that, after activation, a significant portion of the thick filament cross-bridges rotate towards the actin filament resulting in redistribution of the interfilament mass content. They are also consistent with an average orientation of crossbridges in the overlap region different from that in the nonoverlap region.

Animals↗
PubMed1988

Theory of optical ellipsometric measurements from muscle diffraction studies.

A theory of optical ellipsometry describing the complete phase shift and ellipticity of light diffracted from a single muscle fiber is developed. We show that both the phase shift information, described commonly by the birefringence of the fiber, and the ellipticity information, described by the differential polarizability ratio, are necessary to provide a complete picture of the complex contributions to the total optical anisotropy spectra from a diffraction pattern derived from the striated muscle cell. Both form and intrinsic contributions play significant roles in either the birefringence measurement or the differential field ratio measurement. However, we show that their relative weights in these two measured quantities are different, and measuring both of these parameters is necessary to obtain a more complete assessment of the cross-bridge structure and dynamics. The theoretical results have been tested for three different situations: solvent index matching, passive stretch of a resting fiber, and cross-bridge changes under isometric conditions. Comparisons between experimental data and simple model calculations provide much information regarding cross-bridge orientation and structure.

Animals↗