Multiple Envelope Research Articles

Multi-subtype gp160 DNA immunization induces broadly neutralizing anti-HIV antibodies.

A highly desirable feature for an human immunodeficiency virus type 1 (HIV-1) vaccine is the ability to induce broadly reactive anti-envelope antibodies that can neutralize primary HIV-1 isolates. Two immunizations with an HIV-1 envelope-encoding plasmid together with recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) resulted in high antibody titers in mice. The antibody induction was further enhanced after immunization with genes encoding HIV-1 envelopes originating from subtypes A, B and C. The sera from these animals were able to neutralize A, B and C viral isolates, whereas the sera from animals immunized solely with subtype B DNA neutralized only subtype B virus. The combined DNA vaccine gave serum antibodies with broad recognition of HIV-1 envelope epitopes as determined by peptide mapping. Cell-mediated immunity was not compromised by the increased humoral immunity. This demonstrates the ability of multiple envelope genes to induce the desired antibody response against several subtypes. Moreover, it documents the ability of rGM-CSF to enhance the potency of such a vaccine when given simultaneously. The strategy may be useful for making an HIV vaccine more potent and broadly effective against strains of different clades.

Characterization of cross modulation in multichannel amplifiers using a statistically based behavioral modeling technique

A statistically based modeling technique is developed for characterizing in-band and out-of-band intermodulation and cross-modulation distortions in multichannel amplifier environments. The model is based on a new multiple envelope memoryless behavioral model that captures the black-box characteristics of multichannel amplifiers. A power amplifier with a two code-division multiple-access channel signal is characterized experimentally and verifies the approach.

Analysis of the non-covalent interaction between metal ions and the cysteine-Rich domain of protein kinase C eta by electrospray ionization mass spectrometry

Effect of zinc and other metal ions on the folding of the protein kinase C (PKC) surrogate peptide (PKCη-C1B) was analyzed intact under neutral conditions by electrospray ionization mass spectrometry (ESI–MS). ESI–MS spectrum of 64ZnCl2-folded PKCη-C1B clearly showed that PKCη-C1B coordinates specifically two atoms of zinc, and that the two thiol protons are lost in each zinc ion coordinate center. 113CdCl2-folded PKCη-C1B also showed stoichiometry of two cadmium atoms that was proved by addition of EDTA. The dissociation constants of zinc- and cadmium-folded PKCη-C1B in the phorbol 12,13-dibutyrate binding (PDBu) were similar (0.66 and 0.81 nM) with different Bmax values (46.4 and 71.4%). The difference would reflect higher coordination potency of cadmium ion that was demonstrated by ESI–MS when PKCη-C1B was folded by 1:1 mixture of zinc and cadmium ions. In contrast, 63CuCl2-treated PKCη-C1B did not show any copper-coordinated peak, instead a molecular mass less than 6 mass units smaller than that of apo-PKCη-C1B was observed. The multiple charge mass envelope of copper-treated PKCη-C1B shifted to that of the lower mass charge state like zinc-treated PKCη-C1B. These data suggest that the copper treatment formed three intramolecular S–S bonds to abolish the PDBu binding of PKCη-C1B.

Multiple sclerosis retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation.

A retroviral element (MSRV) defining a family of genetically inherited endogenous retroviruses (HERV-W) has recently been characterized in cell cultures from patients with multiple sclerosis (MS). To address the possible relationship with MS, direct detection of circulating virion RNA was proposed but revealed technically difficult to perform in standardized conditions, in the face of multiple endogenous HERV-W copies. A parallel approach has evaluated MSRV potential pathogenicity in relation to characteristic features of multiple sclerosis, in particular, T-lymphocyte-mediated immunopathology. We report here that MSRV particles induce T-lymphocyte response with a bias in the Vbeta16 chain usage in surface receptor, whatever the HLA DR of the donor. A recombinant MSRV envelope-but not core-protein reproduced similar nonconventional activation. Molecular analysis of Vbeta CDR3 showed that Vbeta16 expansions are polyclonal. Our results thus provide evidence that MSRV envelope protein can trigger an abnormal immune response with similar characteristics to that of superantigens.

Primitive, Almost Primitive, Test, and Δ-Primitive Elements of Free Algebras with the Nielsen–Schreier Property

We study generalized primitive elements of free algebras of finite ranks with the Nielsen–Schreier property and their automorphic orbits. A primitive element of a free algebra is an element of some free generating set of this algebra. Almost primitive elements are not primitive elements which are primitive in any proper subalgebra. Δ-primitive elements are elements whose partial derivatives generate the same one-sided ideal of the universal multiplicative envelope algebra of a free algebra as the set of free generators generate. We prove that an endomorphism preserving an automorphic orbit of a nonzero element of a free algebra of rank two is an automorphism. An algorithm to determine test elements of free algebras of rank two is described. A series of almost primitive elements is constructed and new examples of test elements are given. We prove that if the rank n of the free Lie algebra L is even, n=2m, then any Δ-primitive element of L is an automorphic image of the element w=[x1,x2]+···+[x2m−1,x2m], there are no Δ-primitive elements of L if n is odd, and the group of automorphisms of the algebra L acts transitively on the set of all Δ-primitive elements.

Multi-envelope HIV vaccine safety and immunogenicity in small animals and chimpanzees.

A significant obstacle to HIV vaccine development lies in the remarkable diversity of envelope proteins, the major targets of neutralizing antibody. That envelope diversity must be targeted is demonstrated by results from nonhuman primate studies in which single-envelope vaccines have protected against homologous, but rarely against heterologous virus challenges. Similarly, in clinical trials, single-envelope vaccines have failed to prevent break-through infections when challenge viruses were inevitably mismatched with the vaccine. To protect humans from infection by any isolate of HIV, we have prepared vaccine cocktails combining multiple envelopes from distinct viral isolates. We have tested several vehicles for vaccine delivery in small animals and have shown that successive immunizations with envelope, presented first as a DNA recombinant, then as a vaccinia virus (VV) recombinant, and finally as purified protein elicited strong neutralizing antibody responses. We have also tested the VV recombinant vaccine in chimpanzees. Pairs of animals received either single- or multi-envelope VV recombinant vaccines administered by the subcutaneous route. Results showed that the multi-envelope vaccine was safe, immunogenic, and superior to the single-envelope vaccine in eliciting HIV-specific antibody measurable in a standard clinical, immune assay. The promise of this system has led to the initiation of clinical trials, with which the hypothesis that cocktail vaccines will prevent human HIV infections may ultimately be tested.

Rate and Equilibrium Constants for Protein Unfolding and Refolding Determined by Hydrogen Exchange-Mass Spectrometry

Studies of protein unfolding and refolding may help us understand the more general problem of protein folding. Recent studies from this laboratory demonstrated that the unfolding and refolding of a large protein, rabbit muscle aldolase (Mr 157 kDa), can be studied by combining amide hydrogen exchange and mass spectrometry. Results of these studies indicated that aldolase has three unfolding domains which likely unfold sequentially. Urea was used to increase the populations of partially unfolded states which were labeled with deuterium following a brief exposure to D2O. Electrospray ionization mass spectra of both the intact protein and its peptic fragments had multiple envelopes of isotope peaks from which the populations of unfolded forms were determined. The present study extends the previous investigations to include different urea concentrations and kinetic modeling of data taken as the system approaches equilibrium. Analysis of these results gives rate and equilibrium constants describing the unfolding and refolding processes characteristic of aldolase destabilized in urea. The change in solvent-accessible surface, which has been used as a reaction coordinate for protein folding, is estimated from the dependence of the equilibrium and rate constants on the concentration of urea.

Solid-phase synthesis, mass spectrometric analysis of the zinc-folding, and phorbol ester-binding studies of the 116-mer peptide containing the tandem cysteine-rich C1 domains of protein kinase C gamma

Tumor-promoting phorbol esters activate protein kinase C (PKC) isozymes by binding to the zinc-finger like cysteine-rich domains in the N-terminal regulatory region. Our recent studies have revealed that only PKCγ has two high affinity phorbol ester-binding domains, providing a structural blueprint for the rational design of PKCγ-selective modulators for the treatment of neuropathic pain. To extend this approach, the 116-mer peptide containing the double cysteine-rich motifs of PKCγ (γ-C1A-C1B) has been synthesized for the first time using an Fmoc-solid phase strategy with a stepwise chain elongation. This peptide was purified by the reversed phase HPLC to give satisfactory mass data (MALDI-TOF-MS and ESI-TOF-MS). The peptide was successfully folded by zinc treatment and the folded peptide was analyzed intact under neutral conditions by ESI-TOF-MS. The multiple charge mass envelopes shifted to those of the lower mass charge state by addition of 4 molar equiv. ZnCl 2, suggesting that γ-C1A-C1B preserves some higher order structure by the zinc folding. Moreover, the mass spectrum of the zinc-folded peptide in the presence of EDTA clearly showed that γ-C1A-C1B coordinates exactly four atoms of zinc. This zinc stoichiometry is identical to that of native PKCγ. Scatchard analysis of the zinc-folded peptide revealed two binding sites of distinctly different affinities ( K d=6.0±1.5 and 47.0±6.6 nM) comparable to those reported by Quest and Bell for the GST fusion protein of γ-C1A-C1B prepared by DNA recombination. These results indicate that γ-C1A-C1B serves as an effective surrogate for native PKCγ for the study of the structural characteristics of the binding–recognition event and the design, discovery, and development of new PKCγ-selective modulators.

Comodulation masking release (CMR) for multiple temporal envelope maskers

Thresholds for a pure-tone signal were measured in masking noise composed of multiple noise bands. In some conditions, one group of flanking bands (consisting of half of the flanking bands) was comodulated, and independent of the other group of comodulated flanking bands in terms of temporal envelope. The center frequencies of the flanking bands were arranged such that noise bands from the two groups were spectrally interleaved. In these conditions, the band of noise centered on the signal was a combination of two noise bands, which were each comodulated with one group of flanking bands. CMR was obtained but was not of the magnitude observed when all noise bands shared a common temporal envelope. Potential cues in the multiple envelope maskers included the information in the summed representation of the flanking bands, and the degree of temporal envelope correlation between the on-signal band and the flankers. Additional stimulus conditions examined the viability of these cues. The results of these experiments support an envelope correlation explanation for the CMR observed with the multiple temporal envelope maskers. [Work supported by the Louisiana Board of Regents.]

Enhancement of HIV type 1 vaccine immunogenicity by block copolymer adjuvants. I. Induction of high-titer, long-lasting, cross-reactive antibodies of broad isotype.

Improvements in HIV-1 vaccines are urgently needed since many of the available vaccines are weak immunogens. We examined the ability of CRL1005, a novel nonionic block copolymer adjuvant, to improve the immunogenicity of multiple HIV-1 envelope vaccines: six gp120s and single and multiple V3 peptides (MAPs). Formulation of vaccine with adjuvant, as compared with alum or saline, enhanced antibody titer in mice up to 200-fold, with antibody half-lives of >200 days. For most vaccinations, an oil-in-water formulation induced the highest antibody titers; for some antigens, however, particularly single peptides, water-in-oil (w/o) was better. Antigen cross-reactivity was optimized by formulation in w/o, while addition of detoxified lipopolysaccharide enhanced levels of IgG2a and IgG2b. After more than 1 year of observation, no vaccine-related toxicity was observed and emulsified antigen in encapsulated depots was found at immunization sites of w/o-immunized animals. No other adjuvant has been reported to induce such long-lasting antibodies, and the ability of CRL1005 to greatly amplify and qualitatively modify antibody responses suggests that it may be useful in developing improved HIV vaccines for humans.

Ionization within a Cylindrical Capacitor: Electrospray without an Externally Applied High Voltage

An interface was developed for mass spectrometry based on dc induction. Ions were produced as liquid flowed through a concentric cylindrical capacitor at room temperature. No externally applied high voltages affecting the spray tip were used, thus eliminating corona discharge problems in negative ion mode. The voltages applied to the capacitor were lower than those used for electrospray on the same instrument. Stable signals were observed for positive ions infused at flow rates from 50 nL/min to 200 μL/min, anions in the range 50−500 nL/min, without addition of sheath liquid or gas. The minimum concentration required to generate an interpretable product ion spectrum from the [M − 2H]2- precursor, for infused Salmonella typhimurium diphosphorylated lipid A, was improved by a factor of ∼500. Positive ion capillary LC/MS results for a horse apomyoglobin tryptic digest were similar to those observed for an optimized electrospray interface. Factor XIII b subunit, an 83-kDa protein, yielded an interpretable positive ion multiple charge envelope, under buffer conditions that did not allow collection of useful data with our low-flow electrospray source. Mass spectra were also generated for a single-stranded DNA 15-mer and 2‘,4‘-dideuteriotrifluoropropranolol, a small molecule that had been difficult to ionize by either electrospray or APCI.

Enhancement of human immunodeficiency virus type 1 envelope-mediated fusion by a CD4-gp120 complex-specific monoclonal antibody.

The entry of human immunodeficiency virus type 1 (HIV-1) into cells is initiated by binding of the viral glycoprotein gp120-gp41 to its cellular receptor CD4. The gp120-CD4 complex formed at the cell surface undergoes conformational changes that may allow its association with an additional membrane component(s) and the eventual formation of the fusion complex. These conformational rearrangements are accompanied by immunological changes manifested by altered reactivity with monoclonal antibodies specific for the individual components and presentation of new epitopes unique to the postbinding complex. In order to analyze the structure and function of the gp120-CD4 complex, monoclonal antibodies were generated from splenocytes of BALB/c mice immunized with soluble CD4-gp120 (IIIB) molecules (J. M. Gershoni, G. Denisova, D. Raviv, N. I. Smorodinsky, and D. Buyaner, FASEB J. 7:1185-1187 1993). One of those monoclonal antibodies, CG10, was found to be strictly complex specific. Here we demonstrate that this monoclonal antibody can significantly enhance the fusion of CD4+ cells with effector cells expressing multiple HIV-1 envelopes. Both T-cell-line-tropic and macrophage-tropic envelope-mediated cell fusion were enhanced, albeit at different optimal doses. Furthermore, infection of HeLa CD4+ (MAGI) cells by HIV-1 LAI, ELI1, and ELI2 strains was increased two- to fourfold in the presence of CG10 monoclonal antibodies, suggesting an effect on viral entry. These findings indicate the existence of a novel, conserved CD4-gp120 intermediate structure that plays an important role in HIV-1 cell fusion.

Resolving multiple protein conformers in equilibrium unfolding reactions: A time-resolved emission spectroscopic (TRES) study of Azurin

Unlike steady-state spectrofluorimetry, time-resolved emission spectroscopy (TRES) can resolve emissions from fluorophores with similar quantum yields and overlapping steady-state emission spectra. Time-resolved emission studies of the protein-intrinsic fluorophore, tryptophan (Trp), can thus potentially be used to examine protein conformational heterogeneity in solution, as well as to investigate the existence of populated intermediate structural states in equilibrium unfolding reactions of single-tryptophan proteins. Here, the single-Trp copper protein, azurin, is examined in various concentrations of guanidine hydrochloride (GdnCl) with its disulphide bond in an intact state. Interestingly, multiple envelopes of Trp emission are observed in all TRES spectra acquired, instead of just two emission envelopes (corresponding to the native and unfolded states) expected from two-state unfolding. These envelopes appear to be centred around the same set of emission wavelengths in different TRES spectra, and only intensities and decay rates vary with the concentration of denaturant used. This suggests that structural states representing different levels of exposure of Trp to the aqueous solvent might, in fact, be populated at equilibrium during the unfolding of azurin by GdnCl.

The Timescale Correlation Method: Distances to Planetary Nebulae with Halos

We present deep narrow-band CCD images of six PNe obtained with the Palomar 5 m telescope in the light of [N II] ?6584 + H? and [O III] ?5007. Several previously undetected structures are found, including faint multiple envelope structures surrounding three of the targets. The inclusion of these sources with data for other multiple envelope PNe published in the literature permit a statistical evaluation of the relationship between PN shells and the thermal pulses of the PN nucleus. In this paper, we investigate the dynamical timescale between successive envelopes, ?dyn, and the nuclear interpulse timescale, ?ip. Our results show that the deviations from the relation ?dyn = ?ip can be explained by the uncertainty in the Shklovsky distance to individual PNe, which is ?dsh/dsh${r sh}$ --> ~2. By imposing the constraint that ?dyn = ?ip, we find that it is possible to derive a PN distance indicator, which we name the timescale correlation distance, dTC. The derivation for dTC is independent of ad hoc and often unsupported assumptions inherent to the Shklovsky method.

The dynamics of PNe halos and the timescale correlation distance

We present deep narrow-band CCD images of four PNe obtained with the Palomar 5-m telescope in the light of [N II]λ6584 + Hα and [O III]λ5007. Several previously undetected structures are found, including faint multiple envelope structures. The inclusion of these sources with data for other multiple envelope PNe published in the literature permit an evaluation of the relationship between PNe ‘shells’ and the thermal pulses of the PN nucleus.

Auditory evoked responses to amplitude modulated stimuli consisting of multiple envelope components

Steady-state auditory-evoked potentials were recorded noninvasively from alert bottlenosed dolphins, Tursiops truncates, using suction cup electrodes placed on the scalp surface. Responses were elicited using continuous acoustic signals consisting of 2, 3, or 4 tones with lowest frequency at 1000 Hz or 5000 Hz, and having a maximum frequency separation of 171 Hz. Due to the interaction of the stimulus tones, the stimulus waveform was comprised of 1 to 6 dominant temporal envelope components. Evoked responses were averaged in the time domain and Fourier transformed for analysis. The spectrum of the averaged evoked potential contained peaks at Fourier components corresponding to all stimulus envelope frequencies. Thus, scalp potentials, representing the synchronized discharge of large neuronal assemblies, followed the low-frequency temporal envelope of the stimulating waveform whether comprised of 1, 3, or 6 dominant envelope components; this envelope following response (EFR) was the dependent variable in all experiments.

Computational analysis of non-Fourier motion

Non-Fourier motion is now commonplace in research on visual motion perception, yet lacks a computational framework. This paper examines this issue based on the observation that many non-Fourier motion stimuli have a simple characterization in the frequency domain, in terms of oriented power distributions that lie along lines (or planes) that do not pass through the origin. This provides a unifying theoretical framework for a very diverse class of non-Fourier phenomena. It also allows us to examine some central issues concerning the computational nature of non-Fourier models, and naturally occurring sources of non-Fourier motion. For example, it is shown that the orientation of power in frequency domain corresponds to the velocity of a multiplicative envelope, and may arise as a restricted form of lighting effects, translucency or occlusion. We also show that both the location and orientation of spectral power may be extracted from the phase and amplitude output of band-pass filters, consonant with existing non-Fourier models.

Auditory-evoked potentials obtained using continuous amplitude-modulated tones

Auditory-evoked potentials (AEPs) recorded from the scalp surface are neurogenic potentials arising from the massed discharge of neural populations in response to acoustic stimuli and provide a ‘‘window’’ on the physiological basis of auditory processing. AEPs, in particular the transient-evoked auditory brain-stem response (ABR) have been widely used in human clinical settings for the evaluation of auditory functioning. Recently, several new techniques using continuous amplitude-modulated tonal stimuli have been investigated. Many of these techniques take advantage of auditory nonlinearities which result in the auditory system acting as an envelope extractor. This envelope following response (EFR), phase locking of neural discharge to waveform periodicities, has been measuring using two-tone, sinusoidal amplitude-modulated, and multitone, multiple envelope components signals. The EFR offers great promise for frequency specific threshold measurements, especially for low-frequency audiometry and for the examination of auditory processing of complex sounds.

Initiation of Human Cytomegalovirus Infection Requires Initial Interaction with Cell Surface Heparan Sulfate

In this report, we demonstrate that the initial event in human cytomegalovirus (HCMV) infection is attachment to extracellular heparan sulfate. Further, this interaction is important for initiation of infection in fibroblast cells. Using microbinding assays to specifically monitor virus attachment as well as plaque titration assays to measure infectivity, we found that heparin competition as well as enzymatic digestion of cells with heparinase blocked virus attachment, initiation of immediate-early gene expression and infectivity. Other major glycosaminoglycans were found not to be involved in HCMV attachment and infectivity. In addition, HCMV was unable to attach to mutant derivatives of Chinese hamster ovary cells deficient in synthesis of heparan sulfate proteoglycans. Basic fibroblast growth factor, which requires initial interaction with extracellular heparin prior to binding to its high affinity receptor, also inhibited HCMV attachment to cells. Time-course experiments revealed that the initial HCMV binding was sensitive to heparin competition (10 μg/ml) or 0.75 M salt washes. The initial heparin-dissociable binding converted rapidly to high affinity (heparin resistant) HCMV attachment. These data suggest that sequential receptor interactions may mediate HCMV adsorption to cells. Heparin affinity chromatography revealed that multiple HCMV envelope glycoproteins, including gB, are capable of binding to heparin.

REPRESENTATIONS OF FINITELY GENERATED JORDAN PI-ALGEBRAS

The following results are obtained: The universal multiplicative envelope of a finitely generated Jordan PI-algebra is a PI-algebra. The upper nilradical of a finitely generated Jordan PI-algebra over a Noetherian ring is nilpotent. Bibliography: 11 titles.