Analysis Of The Structure And Function Of A Timp-1/cd63 Complex And Its Relationship To An Mt1-Mmp/cd63 Complex

The relationship of the extra-cellular matrix (ECM) and secreted proteins to intra-cellular functions has an important impact on disease progression. The tissue inhibitors of metalloproteinases (TIMPs), of which there are four, are known for their broad metalloprotease inhibitory abilities. TIMP-1 has been widely studied as an inhibitor of matrix metalloproteinases (MMPs) and therefore as a molecule that prevents ECM degradation and proteolytic cleavage of extra-cellular molecules, processes thought to be critical for tumor invasion and metastasis. However, TIMP-1 has shown in the clinic to have increased expression in cases of disease progression; especially, it is associated with poor prognosis in many types of cancer. Hence, there may be oncogenic functions of TIMP-1 that drives cancer progression. We have been studying non-MMP-inhibitory functions of TIMP-1 to better understand TIMP-1 as a pleiotropic molecule and its role in disease development. In this area, we and others have shown important signaling cues mediated by TIMP-1 including anti-apoptotic signaling and epithelial-to-mesenchymal transition (EMT) induction; which may play a role in cancer progression. Our current research has included direct protein-protein interaction analysis using protein complementation assay (PCA) of the interaction of TIMP-1 and CD63. CD63 is a tetraspanin, whose major functional properties are not well understood. We have previously identified CD63 as a non-MMP protein and a transmembrane (TM) protein with which TIMP-1 has direct interaction, resulting in intra-cellular signal transduction for cell survival and EMT. We have characterized the interaction between TIMP-1 and CD63 including identification of their respective binding sites. Importantly, we have also demonstrated a robust interaction for TIMP-1 and CD63 at the cell surface in live cells. In addition, we have utilized PCA methodology in studying the relationship of CD63 with an important disease-related MMP, namely membrane type-1-MMP (MT1-MMP). This MMP is known to be upregulated in cancer progression and has been shown to have interaction with CD63 in previous studies. As we did for TIMP-1, we have mapped the interacting sites of MT1-MMP and CD63. As well, to better understand the relationship of TIMP-1 and MT1-MMP to the binding of each other to CD63 we have studied these three proteins together. We observe that TIMP-1 and MT1-MMP are not compatible in CD63 binding but they compete one with another for interaction with CD63. Our study presents a paradigm shift in our understanding of TIMP-1 functions; we show that the C-terminal domain of TIMP-1, independent of its MMP-inhibitory domain, interacts with CD63 and induces intracellular signal transduction. Importantly, these interactions compete for MT1-MMP binding with CD63, known to mediate endocytosis and degradation of MT1-MMP. Thus, TIMP-1 interaction with CD63 results in MT1-MMP stabilization/accumulation on the cell surface, leading to activation of the MT1-MMP/MMP-2 proteolytic cascade. Our finding provides valuable information for the design of therapeutic intervention of TIMP-1\u27s oncogenic activity while preserving its tumor suppressive MMP-inhibitory functions

Development and Deployment of a Bioreactor for the Removal of Sulfate and Manganese from Circumneutral Coal Mine Drainage

Surface mining, in the form of contour mining and mountain-top removal, is a common means for retrieving coal in the Appalachian Coal Belt region of Kentucky. Overburden or excess spoil generated by these two methods is placed in valley fills. Traditionally Constructed fills have been shown to adversely impact headwater ecosystems via stream burial and through alterations to the hydrology, sediment supply, water quality and biological composition of downstream reaches. Mine drainages emanating from the toe of valley fills often contain elevated levels of total dissolved solids and heavy metals. Drainage chemistry from Guy Cove, a valley fill located in eastern Kentucky, exhibited a mean pH of 6.5 and Fe, Mn and SO4 concentrations of 1.5, 14, and 1264 mg L-1, respectively. The objective of this research was to develop an anaerobic bioreactor for the purpose of reducing Mn and SO4 concentrations in the mine drainage. Development began with batch experiments that tested five different organic carbon sources and five different inorganic substrates. A synthetic mine drainage with a pH of 6.2 and Mn and SO4 concentrations of 90 and 1,500 mg/L, respectively, was used in the experiment. Manganese and SO4 removal varied widely between treatment matrices, with removal rates \u3c 10 to 100% for Mn and \u3c 10 to \u3e 80% for SO4. The substrate sources which provided the most treatment were hardwood mulch and biosolids combined with creek sediment. Subsequent experiments were performed using the synthetic mine drainage in small bioreactors (55 liter plastic tanks) filled with creek sediment with either hardwood mulch or biosolids, each replicated three times. Over a 65 day treatment period \u3e 90% of the Mn and 70% of the SO4 was removed. There were no statistical differences between the two organic substrates. Using this information, in-situ bioreactors consisting of two 5,500 liter plastic septic tanks filled with creek sediment, hardwood mulch and manure compost were installed at Guy Cove. Mine drainage was collected in a sump and conveyed through the inline bioreactors by gravity. Gate valves were used to control flow through the bioreactors. After a 10-month monitoring period, the in-situ bioreactors removed 12, 11, and 64% of Mn, SO4 and Fe from the drainage, respectively. Results from the field differed greatly from those observed under controlled laboratory conditions. Efforts to improve the efficiency of the in-situ bioreactors are underway

\u3ci\u3eAmicus Curiae\u3c/i\u3e Brief of the Hon. Judith Fitzgerald (Bankruptcy Judge, Ret.), and Law Professors Pamela Foohey, George Kuney, Robert Lawless, Jonathan Lipson, Bruce A. Markell, Nancy Rapoport, Richard Squire, Ray Warner and Jack Williams, In Support of the Petitioner

Professor Pamela Foohey filed an amicus brief with a group of law professors and a former bankruptcy judge in MOAC Mall Holdings LLC v. Transform Holdco LLC

HST/FOS Eclipse Observations of the Nova-like Cataclysmic Variable UX Ursae Majoris

[abridged abstract] We present and analyze Hubble Space Telescope observations of the eclipsing nova-like cataclysmic variable UX UMa obtained with the Faint Object Spectrograph. Two eclipses each were observed with the G160L grating (covering the ultraviolet waveband) in August of 1994 and with the PRISM (covering the near-ultraviolet to near-infrared) in November of the same year. The system was 50% brighter in November than in August, which, if due to a change in the accretion rate, indicates a fairly substantial increase in Mdot_acc by >~ 50%. Model disk spectra constructed as ensembles of stellar atmospheres provide poor descriptions of the observed post-eclipse spectra, despite the fact that UX UMa's light should be dominated by the disk at this time. Suitably scaled single temperature model stellar atmospheres with T_eff = 12,500-14,500 K actually provide a better match to both the ultraviolet and optical post-eclipse spectra. Evidently, great care must be taken in attempts to derive accretion rates from comparisons of disk models to observations. One way to reconcile disk models with the observed post-eclipse spectra is to postulate the presence of a significant amount of optically thin material in the system. Such an optically thin component might be associated with the transition region (``chromosphere'') between the disk photosphere and the fast wind from the system, whose presence has been suggested by Knigge & Drew (1997).Comment: 35 pages, including 12 figures; to appear in the ApJ (Vol. 499

Spin Dynamics of the LAGEOS Satellite in Support of a Measurement of the Earth's Gravitomagnetism

LAGEOS is an accurately-tracked, dense spherical satellite covered with 426 retroreflectors. The tracking accuracy is such as to yield a medium term (years to decades) inertial reference frame determined via relatively inexpensive observations. This frame is used as an adjunct to the more difficult and data intensive VLBI absolute frame measurements. There is a substantial secular precession of the satellite's line of nodes consistent with the classical, Newtonian precession due to the non-sphericity of the earth. Ciufolini has suggested the launch of an identical satellite (LAGEOS-3) into an orbit supplementary to that of LAGEOS-1: LAGEOS-3 would then experience an equal and opposite classical precession to that of LAGEOS-1. Besides providing a more accurate real-time measurement of the earth's length of day and polar wobble, this paired-satellite experiment would provide the first direct measurement of the general relativistic frame-dragging effect. Of the five dominant error sources in this experiment, the largest one involves surface forces on the satellite, and their consequent impact on the orbital nodal precession. The surface forces are a function of the spin dynamics of the satellite. Consequently, we undertake here a theoretical effort to model the spin ndynamics of LAGEOS. In this paper we present our preliminary results.Comment: 16 pages, RevTeX, LA-UR-94-1289. (Part I of II, postscript figures in Part II

Capsomer Vaccines Protect Mice from Vaginal Challenge with Human Papillomavirus

Capsomers were produced in bacteria as glutathione-S-transferase (GST) fusion proteins with human papillomavirus type 16 L1 lacking the first nine and final 29 residues (GST-HPV16L1Δ) alone or linked with residues 13–47 of HPV18, HPV31 and HPV45 L2 in tandem (GST-HPV16L1Δ-L2x3). Subcutaneous immunization of mice with GST-HPV16L1Δ or GST-HPV16L1Δ-L2x3 in alum and monophosphoryl lipid A induced similarly high titers of HPV16 neutralizing antibodies. GST-HPV16L1Δ-L2x3 also elicited moderate L2-specific antibody titers. Intravaginal challenge studies showed that immunization of mice with GST-HPV16 L1Δ or GST-HPV16L1Δ-L2x3 capsomers, like Cervarix®, provided complete protection against HPV16. Conversely, vaccination with GST-HPV16 L1Δ capsomers failed to protect against HPV18 challenge, whereas mice immunized with either GST-HPV16L1Δ-L2x3 capsomers or Cervarix® were each completely protected. Thus, while the L2-specific response was moderate, it did not interfere with immunity to L1 in the context of GST-HPV16L1Δ-L2x3 and is sufficient to mediate L2-dependent protection against an experimental vaginal challenge with HPV18

Recovery of 29-s Oscillations in HST/FOS Eclipse Observations of the Cataclysmic Variable UX Ursae Majoris

[abridged abstract] Low amplitude (~=0.5%) 29-s oscillations have been detected in HST/FOS eclipse observations of the nova-like cataclysmic variable UX UMa. These are the same dwarf nova-type oscillations that were originally discovered in this system by Warner & Nather in 1972. The oscillations are sinusoidal to within the small observational errors and undergo an approximately -360 degree phase shift during eclipses. Their amplitudes are highest at pre-eclipse orbital phases and exhibit a rather gradual eclipse whose shape is roughly similar to UX~UMa's overall light curve. Oscillation spectra derived from pre-eclipse data segments are extremely blue, whereas mid-eclipse oscillation spectra are much redder. We suggest that the ultimate source of the oscillations is a hot, compact region near disk center, but that a significant fraction of the observed, modulated flux is due to reprocessing of the light emitted by this source in the accretion disk atmosphere. The compact source is occulted at orbital phases near mid-eclipse, leaving only part of the more extended reprocessing region(s) to produce the weak oscillations that persist even at conjunction. The highly sinusoidal oscillation pulse shape does not permit the identification of the compact component in this model with emission produced by a rotating disturbance in the inner disk or in an equatorial boundary layer, and a standard intermediate polar model can also be ruled out. A model invoking magnetically controlled accretion onto differentially rotating WD surface layers may be viable, but needs more theoretical work.Comment: 28 pages, including 9 figures; to appear in the ApJ (Vol. 499