Antiresonance phase shift in strongly coupled cavity QED.

We investigate phase shifts in the strong coupling regime of single-atom cavity quantum electrodynamics. On the light transmitted through the system, we observe a phase shift associated with an antiresonance and show that both its frequency and width depend solely on the atom, despite the strong coupling to the cavity. This shift is optically controllable and reaches 140°--the largest ever reported for a single emitter. Our result offers a new technique for the characterization of complex integrated quantum circuits.

Entanglement of two individual neutral atoms using Rydberg blockade.

We report the generation of entanglement between two individual 87Rb atoms in hyperfine ground states |F=1,M=1> and |F=2,M=2> which are held in two optical tweezers separated by 4 microm. Our scheme relies on the Rydberg blockade effect which prevents the simultaneous excitation of the two atoms to a Rydberg state. The entangled state is generated in about 200 ns using pulsed two-photon excitation. We quantify the entanglement by applying global Raman rotations on both atoms. We measure that 61% of the initial pairs of atoms are still present at the end of the entangling sequence. These pairs are in the target entangled state with a fidelity of 0.75.

Polarization-controlled single photons.

Vacuum-stimulated Raman transitions are driven between two magnetic substates of a 87Rb atom strongly coupled to an optical cavity. A magnetic field lifts the degeneracy of these states, and the atom is alternately exposed to laser pulses of two different frequencies. This produces a stream of single photons with alternating circular polarization in a predetermined spatiotemporal mode. MHz repetition rates are possible as no recycling of the atom between photon generations is required. Photon indistinguishability is tested by time-resolved two-photon interference.

Drastic reduction of plasmon damping in gold nanorods.

The dephasing of particle plasmons is investigated using light-scattering spectroscopy on individual gold nanoparticles. We find a drastic reduction of the plasmon dephasing rate in nanorods as compared to small nanospheres due to a suppression of interband damping. The rods studied here also show very little radiation damping, due to their small volumes. These findings imply large local-field enhancement factors and relatively high light-scattering efficiencies, making metal nanorods extremely interesting for optical applications. Comparison with theory shows that pure dephasing and interface damping give negligible contributions to the total plasmon dephasing rate.

Specific interaction of a novel foamy virus Env leader protein with the N-terminal Gag domain.

Cryoelectron micrographs of purified human foamy virus (HFV) and feline foamy virus (FFV) particles revealed distinct radial arrangements of Gag proteins. The capsids were surrounded by an internal Gag layer that in turn was surrounded by, and separated from, the viral membrane. The width of this layer was about 8 nm for HFV and 3.8 nm for FFV. This difference in width is assumed to reflect the different sizes of the HFV and FFV MA domains: the HFV MA domain is about 130 residues longer than that of FFV. The distances between the MA layer and the edge of the capsid were identical in different particle classes. In contrast, only particles with a distended envelope displayed an invariant, close spacing between the MA layer and the Env membrane which was absent in the majority of particles. This indicates a specific interaction between MA and Env at an unknown step of morphogenesis. This observation was supported by surface plasmon resonance studies. The purified N-terminal domain of FFV Gag specifically interacted with synthetic peptides and a defined protein domain derived from the N-terminal Env leader protein. The specificity of this interaction was demonstrated by using peptides varying in the conserved Trp residues that are known to be required for HFV budding. The interaction with Gag required residues within the novel virion-associated FFV Env leader protein of about 16.5 kDa.

Organization of immature human immunodeficiency virus type 1.

Immature retrovirus particles contain radially arranged Gag polyproteins in which the N termini lie at the membrane and the C termini extend toward the particle's center. We related image features to the polyprotein domain structure by combining mutagenesis with cryoelectron microscopy and image analysis. The matrix (MA) domain appears as a thin layer tightly associated with the inner face of the viral membrane, separated from the capsid (CA) layer by a low-density region corresponding to its C terminus. Deletion of the entire p6 domain has no effect on the width or spacing of the density layers, suggesting that p6 is not ordered in immature human immunodeficiency virus type 1 (HIV-1). In vitro assembly of a recombinant Gag polyprotein containing only capsid (CA) and nucleocapsid (NC) domains results in the formation of nonenveloped spherical particles which display two layers with density matching that of the CA-NC portion of immature HIV-1 Gag particles. Authentic, immature HIV-1 displays additional surface features and an increased density between the lipid bilayers which reflect the presence of gp41. The other internal features match those of virus-like particles.

Dynamics of serum interleukin-6 level in patients with acute myocardial infarction.

Background: Interleukin-6 (IL-6) is a key cytokine in the initiation of the acute-phase reaction that accompanies myocardial infarction. The study was designed to evaluate changes in serum IL-6 in patients with myocardial infarction and to compare IL-6 alterations with serum creatine kinase (CK) activity. Methods: Serum IL-6 level and CK activity were measured in 19 males with acute myocardial infarction. Blood was taken on the 1st, 3rd, 5th, 7th and 21st days of the disease. Control values were obtained from 30 age-matched healthy males. IL-6 was determined according to the ELISA method and CK was measured with a routine procedure. Results: Serum IL-6 was found to increase on the 1st and 3rd days of the disease, followed by some decline on days 5-21. However, the last values measured were still higher than those in the controls. Patients with transmural myocardial infarction had higher IL-6 levels than those with non-Q myocardial infarction. There was no difference in serum IL-6 in patients with myocardial infarction of the inferior cardiac wall and of the anterior cardiac wall. A correlation was found between IL-6 level and CK activity in the patients, especially on the 1st and 3rd days of the disease. Conclusion: Serum IL-6 increases in patients with myocardial infarction, and this elevation seems to be related to the mass of the affected myocardium. The highest increase is found during the first days of the disease, although the enhanced IL-6 level lasts for at least 3 weeks, probably reflecting the healing process of the myocardium. The clinical value of IL-6 determination does not seem to exceed that of the indices commonly used.

The intact retroviral Env glycoprotein of human foamy virus is a trimer.

Electron microscopy of negatively stained human foamy virus particles provides direct evidence for the trimeric nature of intact Env surface glycoproteins. Three-dimensional image reconstruction reveals that the Env trimer is a tapering spike 14 nm in length. The spikes were often arranged in hexagonal rings which shared adjacent Env trimers.

A conformational switch controlling HIV-1 morphogenesis.

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) proceeds in two steps. Initially, an immature virus with a spherical capsid shell consisting of uncleaved Gag polyproteins is formed. Extracellular proteolytic maturation causes rearrangement of the inner virion structure, leading to the conical capsid of the infectious virus. Using an in vitro assembly system, we show that the same HIV-1 Gag-derived protein can form spherical particles, virtually indistinguishable from immature HIV-1 capsids, as well as tubular or conical particles, resembling the mature core. The assembly phenotype could be correlated with differential binding of the protein to monoclonal antibodies recognizing epitopes in the HIV-1 capsid protein (CA), suggesting distinct conformations of this domain. Only tubular and conical particles were observed when the protein lacked spacer peptide SP1 at the C-terminus of CA, indicating that SP1 may act as a molecular switch, whose presence determines spherical capsid formation, while its cleavage leads to maturation.

Towards the structure of the human immunodeficiency virus: divide and conquer.

Recent publications have expanded our knowledge of the major structural proteins of the human immunodeficiency virus as isolated proteins. The next challenge lies in understanding the changes in structure and the interactions of these components during assembly and maturation.

In vitro assembly properties of wild-type and cyclophilin-binding defective human immunodeficiency virus capsid proteins in the presence and absence of cyclophilin A.

The cellular protein cyclophilin A (CypA) binds specifically to the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein and is incorporated into HIV-1 particles at a molar ratio of 1:10 (CypA/CA). Structural analysis of a CA-CypA complex suggested that CypA may destabilize interactions in the viral capsid and thus promote uncoating. We analyzed the influence of CypA on the in vitro assembly properties of wild-type (WT) CA and derivatives containing substitutions of Gly89 in the Cyp-binding loop. All variant proteins were significantly impaired in CypA binding. In the presence of CypA at a molar ratio of 1:10 (CypA/CA), WT CA assembled into hollow cylinders that were similar to those observed in the absence of CypA but slightly longer. Higher CypA concentrations inhibited cylinder formation. Variant CA proteins G89L and G89F yielded similar cylinders as the WT protein but were significantly more resistant to CypA. Cryoelectron microscopic analysis of WT cylinders assembled in the presence of CypA revealed direct binding of CypA to the outer surface. Electron diffraction patterns generated from these cylinders indicated that CypA causes local disorder. The addition of CypA to preassembled cylinders had little effect, however, and cylinders were only disrupted when incubated with a threefold molar excess of CypA for several hours. These results suggest that CypA does not efficiently destabilize CA interactions at the molar ratio observed in the virion and therefore is unlikely to serve as an uncoating factor.

Actin associates with the nucleocapsid domain of the human immunodeficiency virus Gag polyprotein.

Recently, it was shown that actin molecules are present in human immunodeficiency virus type 1 (HIV-1) particles. We have examined the basis for incorporation and the location of actin molecules within HIV-1 and murine retrovirus particles. Our results show that the retroviral Gag polyprotein is sufficient for actin uptake. Immunolabeling studies demonstrate that actin molecules localize to a specific radial position within the immature particle, clearly displaced from the matrix domain underneath the viral membrane but in proximity to the nucleocapsid (NC) domain of the Gag polyprotein. When virus or subviral Gag particles were disrupted with nonionic detergent, actin molecules remained associated with the disrupted particles. Actin molecules remained in a stable complex with the NC cleavage product (or an NC-RNA complex) after treatment of the disrupted HIV-1 particles with recombinant HIV-1 protease. In contrast, matrix and capsid molecules were released. The same result was obtained when mature HIV-1 particles were disrupted with detergent. Taken together, these results indicate that actin molecules are associated with the NC domain of the viral polyprotein.

Influenza viruses select ordered lipid domains during budding from the plasma membrane.

During the budding of enveloped viruses from the plasma membrane, the lipids are not randomly incorporated into the envelope, but virions seem to have a lipid composition different from the host membrane. Here, we have analyzed lipid assemblies in three different viruses: fowl plague virus (FPV) from the influenza virus family, vesicular stomatitis virus (VSV), and Semliki Forest virus (SFV). Analysis of detergent extractability of proteins, cholesterol, phosphoglycerolipids, and sphingomyelin in virions showed that FPV contains high amounts of detergent-insoluble complexes, whereas such complexes are largely absent from VSV or SFV. Cholesterol depletion from the viral envelope by methyl-beta-cyclodextrin results in increased solubility of sphingomyelin and of the glycoproteins in the FPV envelope. This biochemical behavior suggests that so-called raft-lipid domains are selectively incorporated into the influenza virus envelope. The "fluidity" of the FPV envelope, as measured by the fluorescence polarization of diphenylhexatriene, was significantly lower than compared with VSV or SFV. Furthermore, influenza virus hemagglutinin incorporated into the envelope of recombinant VSV was largely detergent-soluble, indicating the depletion of raft-lipid assemblies from this membrane. The results provide a model for lipid selectivity during virus budding and support the view of lipid rafts as cholesterol-dependent, ordered domains in biological membranes.

The carboxy-terminal p3Gag domain of the human foamy virus Gag precursor is required for efficient virus infectivity.

Proteolytic processing of foamy virus Gag proteins appears to be different from that of other retroviruses. A single carboxy-terminal cleavage site is consistently detectable in human foamy virus (HFV) Gag precursor protein p74Gag derived from infected cells and/or purified virus particles. Using a recombinant HFV protease, we have determined the p74Gag cleavage site that results in p70Gag and the carboxy-terminal p3Gag (Pfrepper et al., 1997, Biochem. Biophys. Res. Commun. 237, 548-553). To study the biological functions of p3Gag, proviral DNA clones were constructed coding for a carboxy-terminally truncated p70Gag lacking the entire p3Gag protein. Removal of p3Gag resulted in an about 100-fold lower virus titer. The expression of other HFV proteins and the processing of Pol proteins were indistinguishable from those of wild-type-transfected cells. The defect in viral infectivity of the p3 mutants was partially restored by coexpressing the full-length p74Gag protein in trans. The deletion of p3Gag resulted in particle assembly with wild-type virion morphology and encapsidation of Pol proteins. Our data show that the carboxy-terminal p3Gag protein has an important function for viral infectivity but is not required for preassembly of capsids, virus morphogenesis, and incorporation of Pol proteins into virions.

Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS and the subject of intense study. The immature HIV-1 particle is traditionally described as having a well ordered, icosahedral structure made up of uncleaved Gag protein surrounded by a lipid bilayer containing envelope proteins. Expression of the Gag protein in eukaryotic cells leads to the budding of membranous virus-like particles (VLPs). RESULTS: We have used cryo-electron microscopy of VLPs from insect cells and lightly fixed, immature HIV-1 particles from human lymphocytes to determine their organization. Both types of particle were heterogeneous in size, varying in diameter from 1200-2600 A. Larger particles appeared to be broken into semi-spherical sectors, each having a radius of curvature of approximately 750 A. No evidence of icosahedral symmetry was found, but local order was evidenced by small arrays of Gag protein that formed facets within the curved sectors. A consistent 270 A radial density was seen, which included a 70 A wide low density feature corresponding to the carboxy-terminal portion of the membrane attached matrix protein and the amino-terminal portion of the capsid protein. CONCLUSIONS: Immature HIV-1 particles and VLPs both have a multi-sector structure characterized, not by an icosahedral organization, but by local order in which the structures of the matrix and capsid regions of Gag change upon cleavage. We propose a model in which lateral interactions between Gag protein molecules yields arrays that are organized into sectors for budding by RNA.

Analysis of vif-defective human immunodeficiency virus type 1 (HIV-1) virions synthesized in 'non-permissive' T lymphoid cells stably infected with selectable HIV-1.

In order to facilitate analyses of the molecular function of the human immunodeficiency virus type 1 (HIV-1) Vif protein, we have developed a cell culture model-system which allows permanent production of genotypically and phenotypically vif-defective HIV-1 virions in 'non-permissive' H9 cells. Using recombinant, replication-competent HIV-1 proviruses coding for a selectable marker gene (gpt) instead of nef, two stably infected H9 subclones named M2 (vif-mutant) and WX (wild-type), respectively, were generated. Virions released from cell line M2--displaying the expected vif-defective phenotype--are produced permanently, and in an at least 50 times higher amount than virus particles from acutely vif-negative HIV-1-infected H9 cells. Analysis of viral protein composition and the electron-microscopic morphology of vif-mutant virions did not reveal any detectable differences in comparison to wild-type virions.

Glycoprotein incorporation and HIV-1 infectivity despite exchange of the gp160 membrane-spanning domain.

We have examined the role of the membrane-anchoring domain of the HIV-1 glycoproteins in viral glycoprotein function, glycoprotein incorporation, and viral infectivity. For this purpose, we initially exchanged the entire membrane-spanning region with that from a cellular glycoprotein (CD22). Subsequently, the strictly conserved arginine in the central position of the transmembranal alpha-helix was replaced by a neutral residue (R696 --> I696). We have further examined the requirements within the cytoplasmic C-terminus for glycoprotein incorporation and replaced this region of gp160 with the long cytoplasmic C-terminus (118 amino acids) from CD22. Our results show that the specific amino acid sequence of the membrane-spanning region of gp160 is not necessary for viral infectivity, thus making it unlikely that this region is specifically involved in membrane fusion, in glycoprotein incorporation, or in infectivity of the cell lines tested. In contrast, recombinant gp160 with the CD22 C-terminal region, although present at the cell surface and membrane fusion-competent, was excluded from incorporation into particles. This could indicate that steric exclusion, and no pseudotyping, occurs when the heterologous, cytoplasmic C-terminal region is too long and not fitting.

Comparison of Seven Commonly Used Agents for Prophylaxis of Seasickness.

The objective of the study was to compare the efficacy and tolerability of seven drugs frequently used for the prevention of seasickness: the drugs were namely cinnarizine, cinnarizine with domperidone, cyclizine, dimenhydrinate with caffeine, ginger root, meclozine with caffeine, and scopolamine. The design was a randomized, double-blind study with two arms. On ethical grounds, a placebo group was not included as in a previous study, in the same setting, 80% of the passengers not receiving prophylactic drugs were seasick. The setting was in Andenes (Norway) during a time period from July to September 1992. Subjects were 1741 tourist volunteers who were joining a whale safari. The main outcome measures were vomiting, malaise (modified Graybiel criteria), and subjective reports of adverse events. Follow up was possible in 1489 volunteers (85.5%). In each active treatment group, 4.1-10.2% experienced vomiting and 16.4-23.5% experienced malaise (not significant). Equally, there was no significant difference in the incidence and characteristics of adverse events reported in the various medication groups. Scopolamine Transdermal Therapeutic System (TTS) users exhibited slightly more visual problems and the agent tended to be less effective. Six of the seven medications may be recommended for prevention of seasickness; scopolamine TTS seems the least attractive.

Expression of biologically active HIV glycoproteins using a T7 RNA polymerase-based eucaryotic vector system.

Bacteriophage T7 RNA polymerase and a derivative containing a nuclear localization signal were transiently expressed in CV-1 cells and were shown to localize to the cytoplasm and nucleus, respectively. A vector was constructed containing T7 promoter and transcription terminator sequences flanking a picornaviral 5' untranslated sequence for cap-independent translation and a polyA signal. Expression of the HIV-1 envelope glycoproteins in this vector system gave high levels of specific transcripts and translation products, independent of the subcellular localization of T7 RNA polymerase. The synthesis of HIV glycoproteins was also completely independent of the coexpression of the HIV rev protein, which is normally required for the expression of HIV structural proteins. In addition, a polyA signal was not required, whereas the presence of the picornaviral 5' untranslated region was necessary for efficient expression. Different possibilities to account for these findings are discussed. The HIV glycoproteins synthesized in this system were normally processed and assembled; they could induce syncytium formation and complement an env-deletion mutant of HIV-1.