Multiplex Zymography Captures Stage-specific Activity Profiles of Cathepsins K, L, and S in Human Breast, Lung, and Cervical Cancer

<p>Abstract</p> <p>Background</p> <p>Cathepsins K, L, and S are cysteine proteases upregulated in cancer and proteolyze extracellular matrix to facilitate metastasis, but difficulty distinguishing specific cathepsin activity in complex tissue extracts confounds scientific studies and employing them for use in clinical diagnoses. Here, we have developed multiplex cathepsin zymography to profile cathepsins K, L, and S activity in 10 μg human breast, lung, and cervical tumors by exploiting unique electrophoretic mobility and renaturation properties.</p> <p>Methods</p> <p>Frozen breast, lung, and cervix cancer tissue lysates and normal organ tissue lysates from the same human patients were obtained (28 breast tissues, 23 lung tissues, and 23 cervix tissues), minced and homogenized prior to loading for cathepsin gelatin zymography to determine enzymatic activity.</p> <p>Results</p> <p>Cleared bands of cathepsin activity were identified and validated in tumor extracts and detected organ- and stage-specific differences in activity. Cathepsin K was unique compared to cathepsins L and S. It was significantly higher for all cancers even at the earliest stage tested (stage I for lung and cervix (n = 6, p < .05), and stage II for breast; n = 6, p < .0001). Interestingly, cervical and breast tumor cathepsin activity was highest at the earliest stage we tested, stages I and II, respectively, and then were significantly lower at the latest stages tested (III and IV, respectively) (n = 6, p < 0.01 and p < 0.05), but lung cathepsin activity increased from one stage to the next (n = 6, p < .05). Using cathepsin K as a diagnostic biomarker for breast cancer detected with multiplex zymography, yielded 100% sensitivity and specificity for 20 breast tissue samples tested (10 normal; 10 tumor) in part due to the consistent absence of cathepsin K in normal breast tissue across all patients.</p> <p>Conclusions</p> <p>To summarize, this sensitive assay provides quantitative outputs of cathepsins K, L, and S activities from mere micrograms of tissue and has potential use as a supplement to histological methods of clinical diagnoses of biopsied human tissue.</p

Overexpression of cathepsin K in mice decreases collagen deposition and lung resistance in response to bleomycin-induced pulmonary fibrosis

<p>Abstract</p> <p>Background</p> <p>Lung fibrosis is a devastating pulmonary disorder characterized by alveolar epithelial injury, extracellular matrix deposition and scar tissue formation. Due to its potent collagenolytic activity, cathepsin K, a lysosomal cysteine protease is an interesting target molecule with therapeutic potential to attenuate bleomycin-induced pulmonary fibrosis in mice. We here tested the hypothesis that over-expression of cathepsin K in the lungs of mice is protective in bleomycin-induced pulmonary fibrosis.</p> <p>Methods</p> <p>Wild-type and cathepsin K overexpressing (cathepsin K transgenic; cath K tg) mice were challenged intratracheally with bleomycin and sacrificed at 1, 2, 3 and 4 weeks post-treatment followed by determination of lung fibrosis by estimating lung collagen content, lung histopathology, leukocytic infiltrates and lung function. In addition, changes in cathepsin K protein levels in the lung were determined by immunohistochemistry, real time RT-PCR and western blotting.</p> <p>Results</p> <p>Cathepsin K protein levels were strongly increased in alveolar macrophages and lung parenchymal tissue of mock-treated cathepsin K transgenic (cath K tg) mice relative to wild-type mice and further increased particularly in cath K tg but also wild-type mice in response to bleomycin. Moreover, cath K tg mice responded with a lower collagen deposition in their lungs, which was accompanied by a significantly lower lung resistance (R_L) compared to bleomycin-treated wild-type mice. In addition, cath K tg mice responded with a lower degree of lung fibrosis than wild-type mice, a process that was found to be independent of inflammatory leukocyte mobilization in response to bleomycin challenge.</p> <p>Conclusion</p> <p>Over-expression of cathepsin K reduced lung collagen deposition and improved lung function parameters in the lungs of transgenic mice, thereby providing at least partial protection against bleomycin-induced lung fibrosis.</p

Visual, Motor and Attentional Influences on Proprioceptive Contributions to Perception of Hand Path Rectilinearity during Reaching

We examined how proprioceptive contributions to perception of hand path straightness are influenced by visual, motor and attentional sources of performance variability during horizontal planar reaching. Subjects held the handle of a robot that constrained goal-directed movements of the hand to the paths of controlled curvature. Subjects attempted to detect the presence of hand path curvature during both active (subject driven) and passive (robot driven) movements that either required active muscle force production or not. Subjects were less able to discriminate curved from straight paths when actively reaching for a target versus when the robot moved their hand through the same curved paths. This effect was especially evident during robot-driven movements requiring concurrent activation of lengthening but not shortening muscles. Subjects were less likely to report curvature and were more variable in reporting when movements appeared straight in a novel “visual channel” condition previously shown to block adaptive updating of motor commands in response to deviations from a straight-line hand path. Similarly, compromised performance was obtained when subjects simultaneously performed a distracting secondary task (key pressing with the contralateral hand). The effects compounded when these last two treatments were combined. It is concluded that environmental, intrinsic and attentional factors all impact the ability to detect deviations from a rectilinear hand path during goal-directed movement by decreasing proprioceptive contributions to limb state estimation. In contrast, response variability increased only in experimental conditions thought to impose additional attentional demands on the observer. Implications of these results for perception and other sensorimotor behaviors are discussed

A Cellular Pathway Involved in Clara Cell to Alveolar Type II Cell Differentiation after Severe Lung Injury

Regeneration of alveolar epithelia following severe pulmonary damage is critical for lung function. We and others have previously shown that Scgb1a1-expressing cells, most likely Clara cells, can give rise to newly generated alveolar type 2 cells (AT2s) in response to severe lung damage induced by either influenza virus infection or bleomycin treatment. In this study, we have investigated cellular pathway underlying the Clara cell to AT2 differentiation. We show that the initial intermediates are bronchiolar epithelial cells that exhibit Clara cell morphology and express Clara cell marker, Scgb1a1, as well as the AT2 cell marker, pro-surfactant protein C (pro-SPC). These cells, referred to as pro-SPC[superscript +] bronchiolar epithelial cells (or SBECs), gradually lose Scgb1a1 expression and give rise to pro-SPC[superscript +] cells in the ring structures in the damaged parenchyma, which appear to differentiate into AT2s via a process sharing some features with that observed during alveolar epithelial development in the embryonic lung. These findings suggest that SBECs are intermediates of Clara cell to AT2 differentiation during the repair of alveolar epithelia following severe pulmonary injury.Singapore-MIT Alliance for Research and Technology Center. Infectious Disease Research Grou

Dusty Planetary Systems

Extensive photometric stellar surveys show that many main sequence stars show emission at infrared and longer wavelengths that is in excess of the stellar photosphere; this emission is thought to arise from circumstellar dust. The presence of dust disks is confirmed by spatially resolved imaging at infrared to millimeter wavelengths (tracing the dust thermal emission), and at optical to near infrared wavelengths (tracing the dust scattered light). Because the expected lifetime of these dust particles is much shorter than the age of the stars (>10 Myr), it is inferred that this solid material not primordial, i.e. the remaining from the placental cloud of gas and dust where the star was born, but instead is replenished by dust-producing planetesimals. These planetesimals are analogous to the asteroids, comets and Kuiper Belt objects (KBOs) in our Solar system that produce the interplanetary dust that gives rise to the zodiacal light (tracing the inner component of the Solar system debris disk). The presence of these "debris disks" around stars with a wide range of masses, luminosities, and metallicities, with and without binary companions, is evidence that planetesimal formation is a robust process that can take place under a wide range of conditions. This chapter is divided in two parts. Part I discusses how the study of the Solar system debris disk and the study of debris disks around other stars can help us learn about the formation, evolution and diversity of planetary systems by shedding light on the frequency and timing of planetesimal formation, the location and physical properties of the planetesimals, the presence of long-period planets, and the dynamical and collisional evolution of the system. Part II reviews the physical processes that affect dust particles in the gas-free environment of a debris disk and their effect on the dust particle size and spatial distribution.Comment: 68 pages, 25 figures. To be published in "Solar and Planetary Systems" (P. Kalas and L. French, Eds.), Volume 3 of the series "Planets, Stars and Stellar Systems" (T.D. Oswalt, Editor-in-chief), Springer 201

Importance of lysosomal cysteine proteases in lung disease

The human lysosomal cysteine proteases are a family of 11 proteases whose members include cathepsins B, C, H, L, and S. The biology of these proteases was largely ignored for decades because of their lysosomal location and the belief that their function was limited to the terminal degradation of proteins. In the past 10 years, this view has changed as these proteases have been found to have specific functions within cells. This review highlights some of these functions, specifically their roles in matrix remodeling and in regulating the immune response, and their relationship to lung diseases

NAHA, a Novel Hydroxamic Acid-Derivative, Inhibits Growth and Angiogenesis of Breast Cancer In Vitro and In Vivo

BACKGROUND: We have recently synthesized novel N-alkylated amino acid-derived hydroxamate, 2-[Benzyl-(2-nitro-benzenesulfonyl)-amino]-N-hydroxy-3-methyl-N-propyl-butyramide (NAHA). Here, we evaluate the anticancer activity of NAHA against highly invasive human breast cancer cells MDA-MB-231 in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Cell growth was evaluated by MTT and soft agar assays. Protein expression was determined by DNA microarray and Western blot analysis. Metastatic potential was evaluated by cell adhesion, migration, invasion, capillary morphogenesis, and ELISA assays. The anticancer activity in vivo was evaluated in mouse xenograft model. NAHA inhibited proliferation and colony formation of MDA-MB-231 cells together with the down-regulation of expression of Cdk2 and CDC20 proteins. NAHA inhibited cell adhesion, migration, and invasion through the suppression of secretion of uPA. NAHA suppressed secretion of VEGF from MDA-MB-231 cells and inhibited capillary morphogenesis of human aortic endothelial cells (HAECs). Finally, NAHA at 50 mg/kg was not toxic and decreased tumor volume and tumor weight in vivo. This suppression of tumor growth was associated with the inhibition of mitotic figures and induction of apoptosis, and the reduction of CD31 and VEGF positive cells in tumors. CONCLUSION: NAHA could be a novel promising compound for the development of new drugs for the therapy of invasive breast cancers

Search for sterile neutrino mixing in the MINOS long-baseline experiment

A search for depletion of the combined flux of active neutrino species over a 735 km baseline is reported using neutral-current interaction data recorded by the MINOS detectors in the NuMI neutrino beam. Such a depletion is not expected according to conventional interpretations of neutrino oscillation data involving the three known neutrino flavors. A depletion would be a signature of oscillations or decay to postulated noninteracting sterile neutrinos, scenarios not ruled out by existing data. From an exposure of 3.18×1020 protons on target in which neutrinos of energies between ~500¿¿MeV and 120 GeV are produced predominantly as ¿µ, the visible energy spectrum of candidate neutral-current reactions in the MINOS far detector is reconstructed. Comparison of this spectrum to that inferred from a similarly selected near-detector sample shows that of the portion of the ¿µ flux observed to disappear in charged-current interaction data, the fraction that could be converting to a sterile state is less than 52% at 90% confidence level (C.L.). The hypothesis that active neutrinos mix with a single sterile neutrino via oscillations is tested by fitting the data to various models. In the particular four-neutrino models considered, the mixing angles ¿24 and ¿34 are constrained to be less than 11° and 56° at 90% C.L., respectively. The possibility that active neutrinos may decay to sterile neutrinos is also investigated. Pure neutrino decay without oscillations is ruled out at 5.4 standard deviations. For the scenario in which active neutrinos decay into sterile states concurrently with neutrino oscillations, a lower limit is established for the neutrino decay lifetime t3/m3&gt;2.1×10-12¿¿s/eV at 90% C.L