Ultraconfined Plasmonic Hotspots Inside Graphene Nanobubbles.

We report on a nanoinfrared (IR) imaging study of ultraconfined plasmonic hotspots inside graphene nanobubbles formed in graphene/hexagonal boron nitride (hBN) heterostructures. The volume of these plasmonic hotspots is more than one-million-times smaller than what could be achieved by free-space IR photons, and their real-space distributions are controlled by the sizes and shapes of the nanobubbles. Theoretical analysis indicates that the observed plasmonic hotspots are formed due to a significant increase of the local plasmon wavelength in the nanobubble regions. Such an increase is attributed to the high sensitivity of graphene plasmons to its dielectric environment. Our work presents a novel scheme for plasmonic hotspot formation and sheds light on future applications of graphene nanobubbles for plasmon-enhanced IR spectroscopy.

T cell responses in calcineurin A alpha-deficient mice.

We have created embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appeared to be normal in CNA alpha -/- mice. CNA alpha -/- T cells responded normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts had a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remained sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA.

Femtosecond pulse sequences used for optical manipulation of molecular motion.

Optical control over elementary molecular motion is enhanced with timed sequences of femtosecond (10(-15) second) pulses produced by pulse-shaping techniques. Appropriately timed pulse sequences are used to repetitively drive selected vibrations of a crystal lattice, in a manner analogous to repetitively pushing a child on a swing with appropriate timing to build up a large oscillation amplitude. This process corresponds to repetitively "pushing" molecules along selected paths in the lattice. Amplification of selected vibrational modes and discrimination against other modes are demonstrated. Prospects for more extensive manipulation of molecular and collective behavior and structure are clearly indicated.

Femtosecond resolution of soft mode dynamics in structural phase transitions.

The microscopic pathway along which ions or molecules in a crystal move during a structural phase transition can often be described in terms of a collective vibrational mode of the lattice. In many cases, this mode, called a "soft" phonon mode because of its characteristically low frequency near the phase transition temperature, is difficult to characterize through conventional frequency-domain spectroscopies such as light or neutron scattering. A femtosecond time-domain analog of light-scattering spectroscopy called impulsive stimulated Raman scattering (ISRS) has been used to examine the soft modes of two perovskite ferroelectric crystals. The low-frequency lattice dynamics of KNbO(3) and BaTiO(3) are clarified in a manner that permits critical evaluation of microscopic models for their ferroelectric transitions. The results illustrate the advantages of ISRS over conventional Raman spectroscopy of low-frequency, heavily damped soft modes.

FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition.

The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action.

The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein: effect of FKBP-12 on single channel activity of the skeletal muscle ryanodine receptor.

The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of terminal cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992-22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca2+ and ryanodine, inhibition by Mg2+ (mM) and ruthenium red (microM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 microM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.

Isolation of a cDNA encoding a novel human FK506-binding protein homolog containing leucine zipper and tetratricopeptide repeat motifs.

Reduced-stringency PCR was used to isolate a cDNA encoding a novel human FK506-binding protein (FKBP) homolog. The encoded 38-kDa protein (FKBPr38) contains at its N-terminus a domain that is 33% identical to FKBP12. FKBPr38 is a member of a subclass of immunophilins, whose other members include FKBP52 and CyP40 (cyclophilin 40), that contain a three-unit tetratricopeptide repeat (TPR). In addition, FKBPr38 contains a consensus leucine-zipper repeat. The presence of the TPR domain and leucine zipper suggest that FKBPr38 may form homo-multimers or interact with other, as yet unidentified, proteins.

32-Indolyl ether derivatives of ascomycin: three-dimensional structures of complexes with FK506-binding protein.

32-Indole ether derivatives of tacrolimus and ascomycin retain the potent immunosuppressive activity of their parent compounds but display reduced toxicity. In addition, their complexes with the 12-kDa FK506-binding protein (FKBP) form more stable complexes with the protein phosphatase calcineurin, the molecular target of these drugs. We have solved the three-dimensional structures of the FKBP complexes with two 32-indolyl derivatives of ascomycin. The structures of the protein and the macrolide are remarkably similar to those seen in the complexes with tacrolimus and ascomycin. The indole groups project away from the body of the complex, and multiple conformations are observed for the linkage to these groups as well as for a nearby peptide suggesting apparent flexibility in these parts of the structure. Comparison of these structures with that of the ternary complex of calcineurin, FKBP, and tacrolimus suggests that the indole groups interact with a binding site comprising elements of both the calcineurin alpha- and beta-chains and that this interaction is responsible for the increased stability of these complexes.

A tacrolimus-related immunosuppressant with biochemical properties distinct from those of tacrolimus.

BACKGROUND: Tacrolimus (FK506) is an immunosuppressive drug 50-100 times more potent than cyclosporine (CsA), the current mainstay of organ transplant rejection therapy. Despite being chemically unrelated, CsA and tacrolimus exert their immunosuppressive effects through the inhibition of calcineurin (CaN), a critical signaling molecule during T-lymphocyte activation. Although numerous clinical studies have proven the therapeutic efficacy of drugs within this class, tacrolimus and CsA also have a strikingly similar profile of unwanted side effects. METHOD: Our objective has been to identify a less toxic immunosuppressant through the modification of ascomycin (FK520). Quantitative in vitro immunosuppression and toxicity assays have demonstrated (see the accompanying article, p. 18) that we achieved our goal with L-732,531 (indolyl-ascomycin; indolyl-ASC), a 32-O-(1-hydroxyethylindol-5-yl) ascomycin derivative with an improved therapeutic index relative to tacrolimus. RESULTS: We report that the attributes of indolyl-ASC may result from its distinctive biochemical properties. In contrast to tacrolimus, indolyl-ASC binds poorly to FK506 binding protein 12 (FKBP12), the major cytosolic receptor for tacrolimus and related compounds. However, the stability of the interaction between the FKBP12-indolyl-ASC complex and CaN is much greater than that of the FKBP12-tacrolimus complex. These distinguishing properties of indolyl-ASC result in the potent inhibition of CaN within T lymphocytes but may lower the accumulation of the drug at sites of toxicity. CONCLUSIONS: Indolyl-ASC may define those properties needed to increase the therapeutic efficacy of a macrolactam immunoregulant for treating both human autoimmune disease and organ transplant rejection.

The mechanism of action of FK-506 and cyclosporin A.

FK-506 and cyclosporin A (CsA) are potent immunosuppressive agents used clinically to prevent tissue rejection. Interest in the development of more effective immunosuppressive drugs has led to an intense effort toward understanding their biochemical mechanism of action with the result that these compounds have now become powerful tools used in deciphering the signal transduction events in T lymphocyte activation. Although chemically unrelated, FK-506 and CsA exert nearly identical biological effects in cells by inhibiting the same subset of early calcium-associated events involved in lymphokine expression, apoptosis, and degranulation. FK-506 binds to a family of intracellular receptors termed the FK-506 binding proteins (FKBPs). CsA binds to another family of intracellular receptors, the cyclophilins (Cyps), distinct from the FKBPs. The similarities between the mechanisms of action of CsA and FK-506 converge upon the calcium- and calmodulin-dependent serine-threonine protein phosphatase calcineurin (CaN). Both the FKBP/FK-506 complex and the Cyp/CsA complex can bind to calcineurin, thereby inhibiting its phosphatase activity. Calcineurin, a component of the signal transduction pathway resulting in IL-2 expression, catalyzes critical dephosphorylation events required for early lymphokine gene transcription.

Designed ultrafast optical nonlinearity in a plasmonic nanorod metamaterial enhanced by nonlocality.

All-optical signal processing enables modulation and transmission speeds not achievable using electronics alone. However, its practical applications are limited by the inherently weak nonlinear effects that govern photon-photon interactions in conventional materials, particularly at high switching rates. Here, we show that the recently discovered nonlocal optical behaviour of plasmonic nanorod metamaterials enables an enhanced, ultrafast, nonlinear optical response. We observe a large (80%) change of transmission through a subwavelength thick slab of metamaterial subjected to a low control light fluence of 7 mJ cm(-2), with switching frequencies in the terahertz range. We show that both the response time and the nonlinearity can be engineered by appropriate design of the metamaterial nanostructure. The use of nonlocality to enhance the nonlinear optical response of metamaterials, demonstrated here in plasmonic nanorod composites, could lead to ultrafast, low-power all-optical information processing in subwavelength-scale devices.

Surface plasmon interference excited by tightly focused laser beams.

We show that interfering surface plasmon polaritons can be excited with a focused laser beam at normal incidence to a plane metal film. No protrusions or holes are needed in this excitation scheme. Depending on the axial position of the focus, the intensity distribution on the metal surface is either dominated by interferences between counterpropagating plasmons or by a two-lobe pattern characteristic of localized surface plasmon excitation. Our experiments can be accurately explained by use of the angular spectrum representation and provide a simple means for locally exciting standing surface plasmon polaritons.

Surface plasmon rainbow jets.

A new method for optically exciting and visualizing surface plasmons in thin metal films is described. The technique relies on the use of a high-numerical-aperture objective lens to locally launch a broad wavelength spectrum of surface waves and to detect the leaky radiative modes associated with them. We used this approach to obtain a direct visualization of the plasmon intensity distributions, e.g., rainbow jets, and to quantify their propagation lengths throughout the visible spectrum.

Surface plasmon characteristics of tunable photoluminescence in single gold nanorods.

Light emission resulting from two-photon excited gold nanoparticles has been proposed to originate from the radiative decay of surface plasmon resonances. In this vein, we investigated luminescence from individual gold nanorods and found that their emission characteristics closely resemble surface plasmon behavior. In particular, we observed spectral similarities between the scattering spectra of individual nanorods and their photoluminescence emission. We also measured a blueshift of the photoluminescence peak wavelength with decreasing aspect ratio of the nanorods as well as an optically tunable shape-dependent spectrum of the photoluminescence. The emission yield of single nanorods strongly depends on the orientation of the incident polarization consistent with the properties of surface plasmons.

Immunopharmacology of rapamycin.

The potent immunosuppressive drugs FK506 and rapamycin interfere with signal transduction pathways required for T cell activation and growth. The distinct inhibitory effects of these drugs on the T cell activation program are mediated through the formation of pharmacologically active complexes with members of a family of intracellular receptors termed the FK506 binding proteins (FKBPs). The FKBP12.FK506 complex specifically binds to and inhibits calcineurin, a signaling protein required for transcriptional activation of the interleukin (IL)-2 gene in response to T cell antigen receptor engagement. The FKBP12. rapamycin complex interacts with a recently defined target protein termed the mammalian target of rapamycin (mTOR). Accumulating data suggest that mTOR functions in a previously unrecognized signal transduction pathway required for the progression of IL-2-stimulated T cells from G1 into the S phase of the cell cycle. Here we review the immunopharmacology of rapamycin, with particular emphasis on the characterization of mTOR.

Purification and properties of the enzymes from Drosophila melanogaster that catalyze the conversion of dihydroneopterin triphosphate to the pyrimidodiazepine precursor of the drosopterins.

The enzyme system responsible for the conversion of 2-amino-4-oxo-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihyd roptridine triphosphate (dihydroneopterin triphosphate or H2-NTP) to 2-amino-4-oxo-6-acetyl-7,8-dihydro-3H,9H-pyrimido[4,5-b]-[1,4]diazepine (pyrimidodiazepine or PDA), a precursor to the red eye pigments, he drosopterins, has been purified from the heads of Drosophila melanogaster. The PDA-synthesizing system consists of two components, a heat-stable enzyme and a heat-labile enzyme. The heat-stable enzyme can be replaced by sepiapterin synthase A, a previously purified enzyme required for the Mg2+-dependent conversion of H2-NTP to an unstable compound that appears to be 6-pyruvoyltetrahydropterin (pyruvoyl-H4-pterin). The heat-labile enzyme, purified to near-homogeneity and termed PDA synthase (Mr = 48,000), catalyzes the conversion of pyruvoyl-H4-pterin to PDA in a reaction requiring the presence of reduced glutathione. Because PDA is two electrons more reduced than pyruvoyl-H4-pterin, the reducing power required for this transformation is probably supplied by glutathione. The PDA-synthesizing system requires the presence of another thiol-containing compound such as 2-mercaptoethanol when incubation conditions 2-mercaptoethanol is no longer required. Evidence is presented to indicate that the Drosophila eye color mutant, sepia, is missing PDA synthase.

Characterization of an exchange reaction between soluble FKBP-12 and the FKBP.ryanodine receptor complex. Modulation by FKBP mutants deficient in peptidyl-prolyl isomerase activity.

FKBP-12 (FKBP), the soluble receptor for the immunosuppresant drug FK-506, is tightly bound to the calcium release channel (CRC)/ryanodine receptor (RyR) of skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum with a stoichiometry of 4 mol of FKBP per tetrameric RyR complex. FKBP displays cis/trans-peptidyl-prolyl isomerase (PPIase) activity which is inhibited by FK-590 or rapamycin. In skeletal muscle TC, FK-590 or rapamycin binds to and dissociates FKBP from the RyR in a time- and temperature-dependent manner which increases the open probability of the channel. Therefore, the net energized Ca2+ uptake rate of TC vesicles devoid of FKBP is reduced due to the increased leak of Ca2+ from the TC specifically via the RyR, which is reversed upon rebinding of FKBP. Thus, the RyR is modulated by FKBP (Timerman, A. P., Ogunbumni, E., Freund, E. A., Wiederrecht, G., Marks, A. R., and Fleischer, S. (1993) J. Biol. Chem. 268, 22922-22999; Mayrleitner, M., Timerman, A. P., Wiederrecht, G., and Fleischer S. (1994) Cell Calcium 15, 99-108). We now find that FKBP can be displaced from the FKBP.RyR complex by exchange with FKBP in solution. The EC50 for exchange is 0.30 microM for wild type FKBP versus 0.6 to 2.4 microM for three different site-directed mutants that are practically devoid of any measurable PPIase activity. Substitution of wild-type FKBP on the RyR complex with these PPIase-deficient mutants did not alter the Ca2+ flux of TC vesicles, whereas dissociation of FKBP from TC with FK-590 increased the Ca2+ leak rate. Our studies show that, in vivo, the FKBP.RyR complex is in equilibrium with the cytosolic pool of FKBP (approximately 3 microM) and suggest that modulation of the CRC by FKBP is independent of PPIase activity.