Chemotactic Activity and Receptor Binding of Neutrophil Attractant/Activation Protein‐1 (NAP‐1) and Structurally Related Host Defense Cytokines: Interaction of NAP‐2 With the NAP‐1 Receptor

Neutrophil attractant/activation protein‐1 (NAP‐1) has sequence similarity to platelet factor‐4 (PF‐4) and to NAP‐2 (a truncated form of connective tissue activating protein‐Ill [CTAP‐III(des 1–15)]. We compared chemotactic activity for neutrophils of these related proteins. We also included for comparison CTAP‐III, CTAP‐III(des 1–13), the C‐terminal dodecapeptide of PF‐4 [PF‐4(59–70)], and C5a. Chemotactic potency (EC50) was highest for NAP‐1 and C5a. Although chemotactic efficacy (peak percentage of neutrophils migrating) was comparable for C5a, NAP‐1, and NAP‐2, the NAP‐2 response occurred only at concentrations 100‐fold higher than the NAP‐1 EC50 of 10‐8 M. Data for the CTAP‐III proteins confirmed that CTAP‐III is not an attractant and that chemotactic activity appears as a result of cleavage of residues at the N‐terminus to make CTAP‐III(des 1–13) or NAP‐2 [CTAP‐III(des 1–15)]. Chemotactic activity of PF‐4 was low and variable, with no significant response by neutrophils from six of nine subjects. In contrast, PF‐4(59–70) regularly induced high chemotactic responses, although the EC50 of 1.6 × 10‐5 M was 1,000‐fold greater than that of NAP‐1. The binding of fluoresceinated NAP‐1 to neutrophils was inhibited by unlabeled NAP‐1 or NAP‐2 but not by PF‐4 or PF‐4 (59–70). This suggests that NAP‐2 interacts with the neutrophil NAP‐1 receptor. Despite the low chemotactic potency of NAP‐2, it is a potential attractant at sites of injury because of the relatively large amounts of the parent CTAP‐III released from platelets, as indicated by a serum concentration of approximately 10‐6 M.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141697/1/jlb0258.pd

The Stochastic Dynamics of Tethered Microcantilevers in a Viscous Fluid

We explore and quantify the coupled dynamics of a pair of micron scale cantilevers immersed in a viscous fluid that are also directly tethered to one another at their tips by a spring force. The spring force, for example, could represent the molecular stiffness or elasticity of a biomolecule or material tethered between the cantilevers. We use deterministic numerical simulations with the fluctuation-dissipation theorem to compute the stochastic dynamics of the cantilever pair for the conditions of experiment when driven only by Brownian motion. We validate our approach by comparing directly with experimental measurements in the absence of the tether which shows excellent agreement. Using numerical simulations, we quantify the correlated dynamics of the cantilever pair over a range of tether stiffness. Our results quantify the sensitivity of the auto- and cross-correlations of equilibrium fluctuations in cantilever displacement to the stiffness of the tether. We show that the tether affects the magnitude of the correlations which can be used in a measurement to probe the properties of an attached tethering substance. For the configurations of current interest using micron scale cantilevers in water, we show that the magnitude of the fluid coupling between the cantilevers is sufficiently small such that the influence of the tether can be significant. Our results show that the cross-correlation is more sensitive to tether stiffness than the auto-correlation indicating that a two-cantilever measurement has improved sensitivity when compared with a measurement using a single cantilever

In Vivo Identification of Eugenol-Responsive and Muscone-Responsive Mouse Odorant Receptors

Our understanding of mammalian olfactory coding has been impeded by the paucity of information about the odorant receptors (ORs) that respond to a given odorant ligand in awake, freely behaving animals. Identifying the ORs that respond in vivo to a given odorant ligand from among the ∼1100 ORs in mice is intrinsically challenging but critical for our understanding of olfactory coding at the periphery. Here, we report an in vivo assay that is based on a novel gene-targeted mouse strain, S100a5-tauGFP, in which a fluorescent reporter selectively marks olfactory sensory neurons that have been activated recently in vivo. Because each olfactory sensory neuron expresses a single OR gene, multiple ORs responding to a given odorant ligand can be identified simultaneously by capturing the population of activated olfactory sensory neurons and using expression profiling methods to screen the repertoire of mouse OR genes. We used this in vivo assay to re-identify known eugenol- and muscone-responsive mouse ORs. We identified additional ORs responsive to eugenol or muscone. Heterologous expression assays confirmed nine eugenol-responsive ORs (Olfr73, Olfr178, Olfr432, Olfr610, Olfr958, Olfr960, Olfr961, Olfr913, and Olfr1234) and four muscone-responsive ORs (Olfr74, Olfr235, Olfr816, and Olfr1440). We found that the human ortholog of Olfr235 and Olfr1440 responds to macrocyclic ketone and lactone musk odorants but not to polycyclic musk odorants or a macrocyclic diester musk odorant. This novel assay, called the Kentucky in vivo odorant ligand-receptor assay, should facilitate the in vivo identification of mouse ORs for a given odorant ligand of interest

Development and deployment of a precision underwater positioning system for in situ laser Raman spectroscopy in the deep ocean

Author Posting. © The Authors, 2005. This is the author's version of the work. It is posted here by permission of Elsevier B. V. for personal use, not for redistribution. The definitive version was published in Deep Sea Research Part I: Oceanographic Research Papers 52 (2005): 2376-2389, doi:10.1016/j.dsr.2005.09.002.The field of ocean geochemistry has recently been expanded to include in situ laser Raman spectroscopic measurements in the deep ocean. While this technique has proved to be successful for transparent targets, such as fluids and gases, difficulty exists in using deep submergence vehicle manipulators to position and control the very small laser spot with respect to opaque samples of interest, such as many rocks, minerals, bacterial mats, and seafloor gas hydrates. We have developed, tested, and successfully deployed by remotely operated vehicle (ROV) a precision underwater positioner (PUP) which provides the stability and precision movement required to perform spectroscopic measurements using the Deep Ocean In Situ Spectrometer (DORISS) instrument on opaque targets in the deep ocean for geochemical research. The positioner is also adaptable to other sensors, such as electrodes, which require precise control and positioning on the seafloor. PUP is capable of translating the DORISS optical head with a precision of 0.1 mm in three dimensions over a range of at least 15 cm, at depths up to 4000 m, and under the normal range of oceanic conditions (T, P, current velocity). The positioner is controlled, and spectra are obtained, in real time via Ethernet by scientists aboard the surface vessel. This capability has allowed us to acquire high quality Raman spectra of targets such as rocks, shells, and gas hydrates on the seafloor, including the ability to scan the laser spot across a rock surface in sub-millimeter increments to identify the constituent mineral grains. These developments have greatly enhanced the ability to obtain in situ Raman spectra on the seafloor from an enormous range of specimens.Funding was provided by a grant to MBARI from the David and Lucile Packard Foundation

Enhanced Damage-Resistant Optics for Spaceflight Laser Systems: Workshop findings and recommendations

NASA has defined a program to address critical laser-induced damage issues peculiar to its remote sensing systems. The Langley Research Center (LaRC), with input from the Goddard Space Flight Center (GSFC), has developed a program plan focusing on the certification of optical materials for spaceflight applications and the development of techniques to determine the reliability of such materials under extended laser exposures. This plan involves cooperative efforts between NASA and optics manufacturers to quantify the performance of optical materials for NASA systems and to ensure NASA's continued application of the highest quality optics possible for enhanced system reliability. A review panel was organized to assess NASA's optical damage concerns and to evaluate the effectiveness of the LaRC proposed program plan. This panel consisted of experts in the areas of laser-induced damage, optical coating manufacture, and the design and development of laser systems for space. The panel was presented information on NASA's current and planned laser remote sensing programs, laser-induced damage problems already encountered in NASA systems, and the proposed program plan to address these issues. Additionally, technical presentations were made on the state of the art in damage mechanisms, optical materials testing, and issues of coating manufacture germane to laser damage

Connective tissue activation

Connective tissue a ctivating p eptides from lymphocytes (CTAP-I) and platelets (CTAP-III) are known to stimulate glycosaminoglycan synthesis, glycolysis, and mitogenesis in connective tissue cell cultures. Direct evidence suggested that increased accumulation of cyclic AMP was involved in the action of these peptide agonists, and increased prostaglandin E synthesis was postulated on the basis of indirect evidence. In the present experiments, CTAP-I and -III were incubated with human and murine cells in culture, and prostaglandin E was measured by radioimmunoassay using antibody directed primarily to prostaglandin E2. Both CTAP-I and -III markedly stimulated the elaboration of prostaglandin E into culture medium, the earliest evidence of increased synthesis occurring at 4 hours with maximal concentrations found at 24 hours. Substantial residual stimulation persisted at least through 48 hours. Indo-methacin (13.0 Mg/ml) obliterated basal and incremental synthesis of prostaglandin in the presence of mediators. Cycloheximide (8.7 Mg/ml) did not affect the stimulation of prostaglandin synthesis by CTAP-I and -III. Three nonrheumatoid and 3 rheumatoid synovial cell strains showed similar basal levels of prostaglandin E and similar responses to CTAP-I. A murine fibroblast cell strain (3T3) showed increased prostaglandin E synthesis on exposure to CTAP-I, and the KB tumor cell strain was markedly stimulated by CTAP-III. These studies confirm the increased synthesis of E series prostaglandins postulated to occur in human connective tissue cells on exposure to CTAP-I and -III, and clarify the mechanism of action of these agonists on “activated” target cells. The importance of elevated extracellular concentrations of prostaglandins is uncertain, although they may act directly on sensitive cell types as well as potentiate the actions of CTAP-I and -III on neighboring cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37752/1/1780240309_ftp.pd