Follicular dendritic cells and the persistence of HIV infectivity: the role of antibodies and Fcgamma receptors

Large quantities of HIV are found trapped on the surface of follicular dendritic cells (FDCs), and virus persists on these cells until they ultimately die. We recently found that FDCs maintain HIV infectivity for long periods in vivo and in vitro. Because FDCs trap Ags (and virus) in the form of immune complexes and are rich in FcgammaRs, we reasoned that Ab and FcgammaRs may be required for FDC-mediated maintenance of HIV infectivity. To investigate this hypothesis, HIV immune complexes were formed in vitro and incubated for increasing times with or without FDCs, after which the remaining infectious virus was determined by HIV-p24 production in rescue cultures. FDCs maintained HIV infectivity in vitro in a dose-dependent manner but required the presence of specific Ab for this activity regardless of whether laboratory-adapted or primary X4 and R5 isolates were tested. In addition, Abs against either virally or host-encoded proteins on the virion permitted FDC-mediated maintenance of HIV infectivity. We found that the addition of FDCs to HIV immune complexes at the onset of culture gave optimal maintenance of infectivity. Moreover, blocking FDC-FcgammaRs or killing the FDCs dramatically reduced their ability to preserve virus infectivity. Finally, FDCs appeared to decrease the spontaneous release of HIV-1 gp120, suggesting that FDC-virus interactions stabilize the virus particle, thus contributing to the maintenance of infectivity. Therefore, optimal maintenance of HIV infectivity requires both Ab against particle-associated determinants and FDC-FcgammaRs.     

Peak capacity of ion mobility mass spectrometry: separation of peptides in helium buffer gas

Advances in the field of proteomics depend upon the development of high-throughput separation methods. Ion mobility-mass spectrometry is a fast separation method (separations on the millisecond time-scale), which has potential for peptide complex mixture analysis. Possible disadvantages of this technique center around the lack of orthogonality between separation based on ion mobility and separation based on mass. In order to examine the utility of ion mobility-mass spectrometry, the peak capacity (phi) of the technique was estimated by subjecting a large dataset of peptides to linear regression analysis to determine an average trend for tryptic peptides. This trend-line, along with the deviation from a linear relationship observed for this dataset, was used to define the separation space for ion mobility-mass spectrometry. Using the maximum deviation found in the dataset (+/-11%) the peak capacity of ion mobility-mass spectrometry is approximately 2600 peptides. These results are discussed in light of other factors that may increase the peak capacity of ion mobility-mass spectrometry (i.e. multiple trends in the data resulting from multiple classes of compounds present in a sample) and current liquid chromatography approaches to complex peptide mixture analysis.     

Beta-cyclodextrin facilitates cholesterol efflux from larval Manduca sexta fat body and midgut in vitro

The ability of 2-hydroxypropyl-β-cyclodextrin (HPβCD) and methyl-β-cyclodextrin (MβCD) to promote cholesterol efflux from [³H]cholesterol-labeled larval Manduca sexta fat body and midgut was tested. In fat body, both β-cyclodextrins induced a two-phase efflux of cholesterol. The first rapid phase depended on cyclodextrin concentration and was more rapid for MβCD than for HPβCD. The second, slower, phase was independent of cyclodextrin concentration and type. In midgut, only the concentration-dependent phase was observed; the rate constants are approximately 85% slower than for fat body. In both cases, a low activation energy for transfer was observed, consistent with a collision mechanism where cyclodextrin interacts directly with cholesterol in plasma membrane to affect transfer. In fat body, the second slower phase is suggestive of a second pool of exchangeable cholesterol and most likely represents transfer of cholesterol from internal membranes or different lateral domains of the plasma membrane. The lack of this second phase in midgut suggests that midgut has only a single pool of exchangeable cholesterol. Although the rates are somewhat different, the overall kinetic pattern for cyclodextrin-mediated cholesterol transfer in insect fat body closely resembles that for vertebrate cells, while the single pool behavior of the midgut is not found in vertebrate cells.     

An analysis of turbulent shear stresses in leakage flow through a bileaflet mechanical prostheses

In this work, estimates of turbulence were made from pulsatile flow laser Doppler velocimetry measurements using traditional phase averaging and averaging after the removal of cyclic variation. These estimates were compared with estimates obtained from steady leakage flow LDV measurements and an analytical method. The results of these studies indicate that leakage jets which are free and planar in shape may be more unstable than other leakage jets, and that cyclic variation does not cause a gross overestimation of the Reynolds stresses at large distances from the leakage jet orifice.     

Mechanistic insights into Sky1p,a yeast homologue of the mammalian SR protein kinases

The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods [Nolen et al. (2001) Nat. Struct. Biol. 8, 176-183]. To determine whether these features impart unique catalytic function, the phosphorylation of the physiological Sky1p substrate, Npl3p, was monitored using steady-state and pre-steady-state kinetic techniques. While Sky1p has a low apparent affinity for ATP compared to other protein kinases, it binds Npl3p with very high affinity. The latter is achieved through a combination of local and distal factors in the protein substrate. The phosphoryl donor ATP has access to the nucleotide pocket in the absence or presence of Npl3p, indicating that a large protein substrate does not enforce an ordered addition of ligands. Sky1p binds two Mg(2+)-the first is essential whereas the second further enhances catalysis. While the turnover number is low (0.5 s(-1)), Npl3p is rapidly phosphorylated in the active site (40 s(-1)) based on single turnover experiments. These results indicate that Sky1p employs a catalytic pathway involving fast phosphoryl transfer followed by slow net release of products. These studies represent the first kinetic investigation of a member of the SRPK family and the first pre-steady-state kinetic study of a protein kinase using a natural protein substrate.     

Stimulus representation in SOP: I. Theoretical rationalization and some implications

THE SOP MODEL [INFORMATION PROCESSING IN ANIMALS: Memory Mechanisms, Erlbaum, Hillsdale, NJ, 1981, p. 5] is described in terms of its assumed stimulus representation, network characteristics, and rules for learning and performance. It is shown how several Pavlovian conditioning phenomena can be accounted on the basis of the model's presumed stimulus representation. Challenges to the SOP model prompted the adoption of a componential stimulus representation in: AESOP [Contemporary Learning Theories: Pavlovian Conditioning and the Status of Traditional Learning Theory, Erlbaum, Hillsdale, NJ, 1989, p. 149], this was a dual representation of the unconditioned stimulus (US), and C-SOP [Contemporary Learning: Theory and Application, Erlbaum, Mahwah, NJ, 2001, p. 23], this was a multi-component representation of the conditioned stimulus (CS). The assumption of a componential CS representation, where large numbers of elements can be separately learned about, necessitated a modification of the learning rule. The modified, "constrained" rule was found useful to explain timing characteristics of Pavlovian conditioned responses, as well as data offered by Rescorla [J. Exp. Psychol. Anim. Behav. Process. 26 (2000) 428; Q. J. Exp. Psychol. 54B (2001) 53; J. Exp. Psychol. Anim. Behav. Process. 28 (2002) 163] showing that stimuli trained in compound do not share the same quantitative fate.     

The Hearing Sensitivity of the Little Skate, Raja erinacea: A Comparison of Two Methods

We determined the hearing sensitivity of the little skate, Raja erinacea using two methods: Behavioral conditioning and the auditory brainstem response (ABR). This marks the first time that the hearing in any member of the Rajiformes has been examined and the first time that the ABR method has been used with an elasmobranch. We obtained audiograms of R. erinacea using each method and were found to be statistically similar. The best hearing sensitivity for R. erinacea was between 100 and 300Hz. We compared the audiograms to audiograms obtained from other species of elasmobranchs. This analysis showed that R. erinacea, a bottom-dwelling elasmobranch, has less sensitive hearing than the lemon shark, Negaprion brevirostris, and the bull shark, Carcharhinus leucas, a free-swimming, raptorial elasmobranch. However, R. erinacea showed sensitivity comparable to that of the horn shark, Heterodontus francisi, another bottom-dwelling elasmobranch; both species feed primarily on benthic prey. These findings are in agreement with Corwin's hypothesis (1978) that hearing sensitivity is correlated with feeding behavior. An examination of the macula neglecta of R. erinacea found a total count of 10000 hair cells, which is within the range of other bottom-dwelling elasmobranchs.     

Stimulus representation in SOP: II. An application to inhibition of delay

The componential extension of SOP accounts for conditioned response (CR) timing in Pavlovian conditioning by assuming that learning accrues with relative independence to stimulus elements that are differentially occasioned during the duration of the conditioned stimulus (CS). SOP, using a competitive learning rule and the assumption that temporal learning emerges via resolution of what is equivalent to an "AX+BX-" discrimination, predicts a progressive increase in the latency of the CR over training, or what Pavlov refer to as "inhibition of delay." Other componential models, which use noncompetitive learning rules, do not predict inhibition of delay. Either type of model makes the prediction indicated, independently of the length of the CS-unconditioned stimulus (US) interval. We report two experiments that demonstrated inhibition of delay when rabbits were trained with relatively long, but not with short, CS-US intervals. To account for this divergence, we assumed that the SOP stimulus trace involves two kinds of elements, some with a temporally distributed pattern of activity over the duration of the CS duration, and some with a randomly distributed pattern. This stimulus representation, not only allows for inhibition of delay with long but not short CS-US intervals, but in combination with SOP's performance rule deduces CR's with "Weber variability."     

Real-time PCR for simultaneous detection and quantification of Borrelia burgdorferi in field-collected Ixodes scapularis ticks from the Northeastern United States

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.