The inhibition by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) of Cl-channels from Torpedo electroplax incorporated in planar phospholipid bilayer membranes is studied. DIDS irreversibly and rapidly inhibits the macroscopic conductance of membranes containing many channels. At the single-channel level, the effect of DIDS is more complicated. The uninhibited single channel displays three "substates" of conductances 20, 10, and 0 pS. Short exposure (5-30 s) to 10 j&M DIDS converts this three-level active channel into a "conventional" channel of 10-pS conductance. Longer exposure eliminates all channel fluctuations. The results are taken as strong evidence that the ClF channel is constructed as a functional dimer of identical protein subunits.In the plasma membrane of the electric organ of Torpedo californica resides a Cl--specific channel whose function is to establish the electroplax cell as a low-internal-resistance battery that the fish may use as a source of electric current in stunning its prey (1-5). This channel has been characterized in detail, after insertion into planar lipid bilayers (1-5) or giant lipid vesicles (6, 7), by observation of current fluctuations under voltage-clamp conditions. At the single-channel level, an unusual gating behavior is observed: a "bursting" process in which the open channel displays three distinguishable "substates" (4,5). This open-channel substructure is uncommonly well-behaved and has led to a simple physical picture of the Cl-channel as a functional dimer. We have proposed that the three active substates result from the independent opening and closing of two identical Cl-diffusion pathways, or "protochannels" (4, 5). In this scheme, the dimeric channel complex may exist with both protochannels simultaneously open, with one open and one closed, or with both closed. In this way, the three substates, equally spaced in conductance, are generated. According to this view, the protochannels must be intimately associated in a complex because of the channel's bursting behavior: the simultaneous appearance and disappearance of both protochannels.This "double-barreled shotgun" model for the channel is quite eccentric, but it is well supported by several lines of evidence (4, 5): the equal spacing in conductance of the three substates, the binomially distributed probabilities of the appearance of the substates, and the "dimeric" transition probabilities among the substates. But this stochastic evidence is, ultimately, indirect. In this report, we present much stronger evidence for the dimeric structure of this channel by examining the action of 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) on the channel's substate behavior. We argue that this irreversible inhibitor can be used to trap a "half-inhibited" channel complex in which only one of the protochannels is able to function and, thus, may be observed directly.MATERIALS AND METHODS Biochemical. Noninnervated face vesicles of T. californica electroplax were prepared immediately after dissection of the tissue...

[1] The NASA Discovery Moon Mineralogy Mapper imaging spectrometer was selected to pursue a wide range of science objectives requiring measurement of composition at fine spatial scales over the full lunar surface. To pursue these objectives, a broad spectral range imaging spectrometer with high uniformity and high signal-to-noise ratio capable of measuring compositionally diagnostic spectral absorption features from a wide variety of known and possible lunar materials was required. For this purpose the Moon Mineralogy Mapper imaging spectrometer was designed and developed that measures the spectral range from 430 to 3000 nm with 10 nm spectral sampling through a 24 degree field of view with 0.7 milliradian spatial sampling. The instrument has a signal-to-noise ratio of greater than 400 for the specified equatorial reference radiance and greater than 100 for the polar reference radiance. The spectral cross-track uniformity is >90% and spectral instantaneous field-of-view uniformity is >90%. The Moon Mineralogy Mapper was launched on Chandrayaan-1 on the 22nd of October. On the 18th of November 2008 the Moon Mineralogy Mapper was turned on and collected a first light data set within 24 h. During this early checkout period and throughout the mission the spacecraft thermal environment and orbital parameters varied more than expected and placed operational and data quality constraints on the measurements. On the 29th of August 2009, spacecraft communication was lost. Over the course of the flight mission 1542 downlinked data sets were acquired that provide coverage of more than 95% of the lunar surface. An end-to-end science data calibration system was developed and all measurements have been passed through this system and delivered to the Planetary Data System (PDS.NASA.GOV). An extensive effort has been undertaken by the science team to validate the Moon Mineralogy Mapper science measurements in the context of the mission objectives. A focused spectral, radiometric, spatial, and uniformity validation effort has been pursued

SummaryFollowing occupancy of the T cell receptor by antigen, T cell proliferation and lymphokine production are determined by a second costimulatory signal delivered by a ligand expressed on antigen presenting cells. The human B cell activation antigen B7, which is expressed on antigen presenting cells including activated B cells and 'Y interferon treated monorytes, has been shown to deliver such a costimulatory signal upon attachment to its ligand on T cells, CD28. We have cloned and sequenced the murine homologue of the human B7 gene . The predicted murine protein has 44% amino acid identity with human B7 . The greatest similarity is in the IgV and Ig-C like domains . Murine B7 mRNA was detected in murine hematopoietic cells of B cell but not T cell origin . Cells transfected with murine B7 provided a costimulatory signal to human CD28+ T lymphocytes. These results demonstrate the costimulatory activity of murine B7 and provide evidence that the ligand attachment site is conserved between the two species .Although occupancy of the TCR complex by antigen in 13, association with the MHC is necessary for the initiation of T cell activation, several lines of evidence suggest that a second costimulatory signal is essential for the induction of proliferation and lymphokine secretion (1-4) . In murine and human systems, this costimulatory signal is delivered by APC and requires cell to cell contact (2, 4) . Cells which can deliver this costimulatory signal include activated, but not resting B lymphocytes (5), INF-y activated monorytes, and dendritic cells (2, 6) .Several recent studies in human systems have provided compelling evidence that the B cell activation antigen B7 can provide one such costimulatory signal (7-9) . B7, a member of the Ig supergene family, has been shown to be a ligand for another member of this family, the T cell surface protein CD28, (10-13) . CD28 is constitutively expressed on 95% of human CD4+ T cells, 50% of CD8+ T cells, and on thymocytes which coexpress CD4 and CD8 (14-16) . Following suboptimal activation of T cells with anti-CD3 mAb (16), anti-CD2 mAb, or phorbol ester (17), crosslinking of CD28 by anti-CD28 mAb results in enhanced T cell proliferation and greatly augments synthesis of multiple lymphokines (18). B7 is likely to be an important regulator of T cell proliferation and lymphokine production as evidenced by its pattern of expression and functional activity. B7 is not expressed on resting B cells (19) but appears following crosslinking of surface Ig (10, 19) or class II MHC (9) . Moreover, B7 is not expressed on unstimulated monorytes and is specifically induced by INF-y but not other stimuli which activate monocytes (20). Human B7 (hB7) 1 transfected cells or recombinant B7-Ig fusion protein augment proliferation and induce IL2, but not IIT4, synthesis in T cells which have been treated with phorbol ester or anti-CD3 mAb (7-9) .In murine systems, a homologue for CD28 has recently been cloned (21); however, a conserved functional activity has not yet been demonstrated . In ...

Novel effects of cholesterol (Chol) on nicotinic acetylcholine receptor (AChR) cell-surface stability, internalization and function are reported. AChRs are shown to occur in the form of submicron-sized (240-280 nm) domains that remain stable at the cell-surface membrane of CHO-K1/A5 cells over a period of hours. Acute (30 min, 37 degrees C) exposure to methyl-beta-cyclodextrin (CDx), commonly used as a diagnostic tool of endocytic mechanisms, is shown here to enhance AChR internalization kinetics in the receptor-expressing clonal cell line. This treatment drastically reduced ( approximately 50%) the number of receptor domains by accelerating the rate of endocytosis (t(1/2) decreased from 1.5-0.5 h). In addition, Chol depletion produced ion channel gain-of-function of the remaining cell-surface AChR, whereas Chol enrichment had the opposite effect. Fluorescence measurements under conditions of direct excitation of the probe Laurdan and of Förster-type resonance energy transfer (FRET) using the intrinsic protein fluorescence as donor both indicated an increase in membrane fluidity in the bulk membrane and in the immediate environment of the AChR protein upon Chol depletion. Homeostatic control of Chol content at the plasmalemma may thus modulate cell-surface organization and stability of receptor domains, and fine tune receptor channel function to temporarily compensate for acute AChR loss from the cell surface.

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