Clues from digital radio regarding biomolecular recognition.

Frequency-offset immunosensors based on acoustic wave devices are known to provide extremely high sensitivity and selectivity where the target is detected and identified based on the amount of frequency shift. We propose a new method to further classify chemically similar molecules extrapolating on the concept of in-phase (I) and quadrature (Q) domain used for the detection of orthogonal M-ary signals in digital telecommunication systems. We performed a series of detection experiments using samples of explosives such as cyclotrimethylene trinitramine [or royal demolition explosive (RDX)] and trinitrotoluene (TNT), containing nitrous oxide (NO2) groups and chemically analogous substances (e.g., musk oil). This detection scheme involves the use of semi-orthogonal monoclonal anti-TNT and anti-RDX antibodies immobilized onto two separate sensor surfaces. The term semi-orthogonal represents the co-option of a term used heavily in digital radio for the purpose of describing chemical orthogonality. The antibody to TNT which we use has some reactivity with RDX, and other nitrous oxide compounds. This feature of an antibody is referred to in the literature as antibody promiscuity. The antibody for RDX which we use shows very little cross reactivity with other molecules and, hence, the chemical responsiveness of the two antibodies is not quite orthogonal. Their responses are then chemically semi-orthogonal. The two semi-orthogonal immunosensor responses were then monitored and the baseline frequency shifts were recorded. After remapping the measured frequency data of the analytes onto a new 2-D domain by setting the TNT-specific sensor as the (I) or real component and the RDX-specific sensor as the (Q) or imaginary component, we could observe that all the substances were detected and mapped out to distinct regions on the I-Q plot. We assert that there is a strong resemblance between digital radio system quadrature detection techniques and our I-Q mapping scheme of the semi-orthogonal immunosensor signatures.

Human immunodeficiency virus type 1 subtype B ancestral envelope protein is functional and elicits neutralizing antibodies in rabbits similar to those elicited by a circulating subtype B envelope.

Human immunodeficiency virus type 1 (HIV-1) is a difficult target for vaccine development, in part because of its ever-expanding genetic diversity and attendant capacity to escape immunologic recognition. Vaccine efficacy might be improved by maximizing immunogen antigenic similarity to viruses likely to be encountered by vaccinees. To this end, we designed a prototype HIV-1 envelope vaccine using a deduced ancestral state for the env gene. The ancestral state reconstruction method was shown to be >95% accurate by computer simulation and 99.8% accurate when estimating the known inoculum used in an experimental infection study in rhesus macaques. Furthermore, the deduced ancestor gene differed from the set of sequences used to derive the ancestor by an average of 12.3%, while these latter sequences were an average of 17.3% different from each other. A full-length ancestral subtype B HIV-1 env gene was constructed and shown to produce a glycoprotein of 160 kDa that bound and fused with cells expressing the HIV-1 coreceptor CCR5. This Env was also functional in a virus pseudotype assay. When either gp160- or gp140-expressing plasmids and recombinant gp120 were used to immunize rabbits in a DNA prime-protein boost regimen, the artificial gene induced immunoglobulin G antibodies capable of weakly neutralizing heterologous primary HIV-1 strains. The results were similar for rabbits immunized in parallel with a natural isolate, HIV-1 SF162. Further design efforts to better present conserved neutralization determinants are warranted.

Modeling a longitudinally coupled dual-mode leaky-SAW resonator filter with enhanced upper-sideband suppression.

Recently, it has been experimentally reported that enhanced upper-sideband suppression of a longitudinally coupled (first/third) dual-mode leaky SAW (LSAW) resonator filter may be obtained by incorporation of a selectively valued capacitor between input and output terminals. In this paper, coupling-of-modes (COM) and bandstop-filter modeling is applied to realize this enhanced suppression. Tradeoffs are examined between upper and lower side-band suppression levels caused by the ensuing capacitive coupling between input and output terminals. Good agreement is obtained between this theory and reported experimental results for an 800-MHz band cellular filter.

Conductance measurements on a leaky SAW harmonic one-port resonator.

Conductance measurements are reported on a leaky SAW (LSAW) harmonic one-port resonator on a 64 degrees Y-X LiNbO(3) substrate. This employed a short three-finger IDT for fundamental and second harmonic operation together with long reflection gratings. Conductances were measured with and without the end gratings. From an analysis of the measurements, it was deduced that, for optimum second harmonic performance, the grating stop-band frequency should be higher than the IDT unperturbed center frequency. This result is in contrast to fundamental frequency resonator designs in which the end grating stop-band frequency is placed below the IDT center frequency for optimum performance.

Evolution of a simian immunodeficiency virus pathogen.

Analysis of disease induction by simian immunodeficiency viruses (SIV) in macaques was initially hampered by a lack of molecularly defined pathogenic strains. The first molecularly cloned SIV strains inoculated into macaques, SIVmacBK28 and SIVmacBK44 (hereafter designated BK28 and BK44, respectively), were cases in point, since they failed to induce disease within 1 year postinoculation in any inoculated animal. Here we report the natural history of infection with BK28 and BK44 in inoculated rhesus macaques and efforts to increase the pathogenicity of BK28 through genetic manipulation and in vivo passage. BK44 infection resulted in no disease in four animals infected for more than 7 years, whereas BK28 induced disease in less than half of animals monitored for up to 7 years. Elongation of the BK28 transmembrane protein (TM) coding sequence truncated by prior passage in human cells marginally increased pathogenicity, with two of four animals dying in the third year and one dying in the seventh year of infection. Modification of the BK28 long terminal repeat to include four consensus nuclear factor SP1 and two consensus NF-kappaB binding sites enhanced early virus replication without augmenting pathogenicity. In contrast, in vivo passage of BK28 from the first animal to die from immunodeficiency disease (1.5 years after infection) resulted in a consistently pathogenic strain and a 50% survival time of about 1.3 years, thus corresponding to one of the most pathogenic SIV strains identified to date. To determine whether the diverse viral quasispecies that evolved during in vivo passage was required for pathogenicity or whether a more virulent virus variant had evolved, we generated a molecular clone composed of the 3' half of the viral genome derived from the in vivo-passaged virus (H824) fused with the 5' half of the BK28 genome. Kinetics of disease induction with this cloned virus (BK28/H824) were similar to those with the in vivo-passaged virus, with four of five animals surviving less than 1.7 years. Thus, evolution of variants with enhanced pathogenicity can account for the increased pathogenicity of this SIV strain. The genetic changes responsible for this virulent transformation included at most 59 point mutations and 3 length-change mutations. The critical mutations were likely to have been multiple and dispersed, including elongation of the TM and Nef coding sequences; changes in RNA splice donor and acceptor sites, TATA box sites, and Sp1 sites; multiple changes in the V2 region of SU, including a consensus neutralization epitope; and five new N-linked glycosylation sites in SU.

Incomplete protection, but suppression of virus burden, elicited by subunit simian immunodeficiency virus vaccines.

We compared the efficacy of immunization with either simian immunodeficiency virus (SIV) Env glycoprotein (Env), Env plus Gag proteins (Gag-Env), or whole inactivated virus (WIV), with or without recombinant live vaccinia vector (VV) priming, in protecting 23 rhesus macaques (six vaccine and two control groups) from challenge with SIVmac251 clone BK28. Vaccination elicited high titers of syncytium-inhibiting and anti-Env (gp120/gp160) antibodies in all vaccinated macaques and anti-Gag (p27) antibodies in groups immunized with WIV or Gag-Env. Only WIV-immunized macaques developed anticell (HuT78) antibodies. After homologous low-dose intravenous virus challenge, we used frequency of virus isolation, provirus burden, and change in antibody titers to define four levels of resistance to SIV infection as follows. (i) No infection ("sterilizing" immunity) was induced only in WIV-immunized animals. (ii) Abortive infection (strong immunity) was defined when virus or provirus were detected early in the postchallenge period but not thereafter and no evidence of virus or provirus was detected in terminal tissues. This response was observed in two animals (one VV-Env and one Gag-Env). (iii) Suppression of infection (incomplete or partial immunity) described a gradient of virus suppression manifested by termination of viremia, declining postchallenge antibody titers, and low levels (composite mean = 9.1 copies per 10(6) cells) of provirus detectable in peripheral blood mononuclear cells or lymphoid tissues at termination (40 weeks postchallenge). This response occurred in the majority (8 of 12) of subunit-vaccinated animals. (iv) Active infection (no immunity) was characterized by persistent virus isolation from blood mononuclear cells, increasing viral antibody titers postchallenge, and high levels (composite mean = 198 copies per 10(6) cells) of provirus in terminal tissues and blood. Active infection developed in all controls and two of three VV-Gag-Env-immunized animals. The results of this study restate the protective effect of inactivated whole virus vaccines produced in heterologous cells but more importantly demonstrate that a gradient of suppression of challenge virus growth, reflecting partial resistance to SIV infection, is induced by subunit vaccination. The latter finding may be pertinent to studies with human immunodeficiency virus vaccines, in which it is plausible that vaccination may elicit significant suppression of virus infection and pathogenicity rather than sterilizing immunity.

Injection locking techniques for a 1-GHz digital receiver using acoustic-wave devices.

The use of surface-transverse-wave (STW) resonator-based oscillators as amplifiers and as carrier recovery elements is discussed. It is demonstrated that these oscillators can amplify phase-shift-keyed signals with very little added noise, while providing a constant output power. Their performance in carrier recovery amplifications is analyzed. Experimental results showing the amplification with more than 80 dB of dynamic range of a 2 Mb/s BPSK signal and the carrier recovery of an 8 Mb/s signal with a 1-GHz STW oscillator are shown.

Aspects of modeling the frequency response of a two-port waveguide-coupled SAW resonator-filter.

Coupling-of-modes-in-time and coupling-of-modes-in-space analyses are applied to the modeling of a two-port two-pole waveguide-coupled SAW resonator filter. Modeling is applied to a lumped equivalent circuit and a distributed circuit. While the accuracy of the coupling estimation dictates the exact mode-splitting and bandwidth, the ensuing computations can give a good representation of the amplitude and phase response, including the effect of degenerate transverse modes.

Transverse modes in one-port SAW resonators.

The S(11) and equivalent S(21) frequency responses of a one-port surface acoustic wave (SAW) resonator with transverse modes derived from one-dimensional coupling-of-modes and transmission-matrix analysis. The two-dimensional nature of the problem is approximated by a summation of one-dimensional mode responses for each transverse mode. Comparison between theory and experimental data for a commercial 280-MHz one-port SAW resonator shows good agreement for the placement of transverse modes.

A surface transverse wave-based MSK system.

A minimum-shift-keyed (MSK) system using one 1-GHz surface-transverse-wave (STW) resonator in the transmitter and two identical STW resonators in the receiver is described. It is shown to operate with data rates of over 100 kb/s and consume 150 mW. The system has a very high sensitivity and is tolerant to additive noise. Since the signal is never up- or down-converted. this system eliminates high power consumption. The system should find applications in cellular radios and in industrial communications.

Herpes simplex virus type 2 mutants with deletions in the intergenic region between ICP4 and ICP22/47: identification of nonessential cis-acting elements in the context of the viral genome.

In herpes simplex virus type 2, the mRNAs of ICP4 and ICP22/47 are divergently transcribed and their transcription initiation sites are separated by 750 base pairs (L. J. Whitton and J. B. Clements, Nucleic Acids Res. 12:2061-2078, 1984). This 750-base-pair region contains many recognized cis-acting elements, including two TATA boxes, numerous Sp1-binding sites, four TAATGARAT motifs, at least one ICP4-binding site, and two origins of replication (oriS) linked in tandem. In this report, we describe the construction of mutant viruses with defined deletions that eliminate these elements either singly or in combination. The phenotypic properties of these mutants indicate that (i) the TAATGARAT motifs and their neighboring elements affect the levels of transcription of both ICP4 and ICP22/47 similarly, (ii) the TATA box serving ICP4 is required for efficient ICP4 mRNA synthesis and for determining the initiation site of transcription, (iii) the ICP4-binding site located at the start of ICP4 transcription is at least partially responsible for the decreased levels of ICP4 mRNA observed in the presence of immediate-early and early gene products, and (iv) mutants bearing deletions that eliminate the entire conventionally recognized ICP4 promoter generate sufficient ICP4 mRNA to maintain viability in cells not expressing ICP4. Additionally, our inability to generate viable deletion mutants lacking all copies of oriS suggests that at least one copy of oriS may be essential for virus replication.

Mode selection in a multimode SAW oscillator using FM chirp mixing signal injection.

Mode-selection control of a multimode surface-acoustic-wave (SAW) oscillator has been obtained using SAW linear FM chirp signal injection. The prototype 60-100-MHz SAW oscillator design employed a single-phase unidirectional transducer (SPUDT) low-loss comb filter in the feedback loop, with minimum insertion loss of approximately 3.7 dB. Mode selection was achieved using an injection signal derived from the mixed output of two 27.5-52.5-MHz up- and down-chirp SAW filters. Mode switching times of less, similar2 mus were obtained. The device could be useful as a local oscillator on frequency-agile radars, where hopping is required over a moderate number of frequencies.

L-Carnitine dissimilation in the gastrointestinal tract of the rat.

Results of previous studies in this laboratory and others have suggested that L-carnitine is degraded in the gastrointestinal tract of the rat, perhaps by the action of indigenous flora. L-[methyl-14C]Carnitine was administered to rats either orally or intravenously in doses of 86 nmol or 124 mumol, and expired air, 48-h urine and fecal collections, and selected tissues at 48 h after isotope administration were examined for radiolabeled carnitine and metabolites. Urine and feces of rats receiving oral L-[methyl-14C]carnitine consistently contained two radiolabeled metabolites which were identified as trimethylamine N-oxide (primarily in urine) and gamma-butyrobetaine (primarily in feces). In these rats, these metabolites accounted for up to 23% and 31% of the administered dose, respectively. By contrast, for rats receiving intravenous L-[methyl-14C]carnitine or germ-free rats receiving the isotope orally or intravenously, virtually all of the radioactivity recovered was in the form of carnitine. Analyses for 14CO2 and [14C]trimethylamine in expired air revealed little or no (less than 0.1% of dose) conversion to these compounds, regardless of size of dose or route of administration. Results of this study demonstrate conclusively that L-carnitine is degraded in the gastrointestinal tract of the rat and that indigenous flora are responsible for these transformations.