Enumeration of lymphokine-secreting cells as a quantitative measure for cellular immune responses in rhesus macaques

Abstract: We investigated whether enumeration of lymphokine-secreting T cells can be used as a quantitative measure to determine the immunogenicity of foreign proteins in rhesus monkeys. In addition, it was assessed whether this approach can supplement and/or substitute for the well-established lymphoproliferation assay. Two candidate vaccine proteins (e.g., HIV-1 gp120 and HSV-2gD) were used as model antigens for immunization. PBMCs from immunized animals were antigenically stimulated and evaluated on their proliferative capacity and lymphokine release at the single cell level. The experiments showed a close quantitative correlation between antigen-triggered proliferative responses and the antigen-induced generation of Il-2 and IFN-gamma producing cells (pc). Il-4pc were found to appear relatively late after the initiation of antigen exposure. The data indicate that ELISPOT assays provide valuable tools for the assessment of the antigenicity of foreign proteins in vivo.     

THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. II. ISOLATION OF A BINDING PROTEIN WHICH PARTIALLY ACTIVATES EGGS1

An in vitro binding assay involving egg plasma membrane vesicles (PMVs) of Fucus serratus L. and proteins contained in a KCl extract of sperm has been used to identify a sperm protein involved in egg binding. High-performance gel filtration (HPGF) separated the sperm KCl extract into several major fractions, and a protein (apparent M, 60 kDa) was identified as being involved in binding to the egg PMVs. This protein ran on denaturing sodium dodecyl sulfate (SDS)gels with an apparent molecular weight of 27 kDa. This suggests that either the native form of the protein is a dimer or the molecular weight on HPGF is an artifact caused by high ionic strength buffer promoting hydrophobic interactions. When KCl-sol-uble proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), blotted onto nitrocellulose, and incubated with biotinylated egg PMVs, these bound to a band at 27 kDa, confirming the role of this protein. Addition of the Fucus sperm extract or HPGF fractions containing the binding protein to eggs in the absence of sperm induced the release of polysaccharides onto the egg cell surface. This labeling was patchy, in contrast to the uniform release of polysaccharides observed when sperm were added to eggs. The monoclonal antibody (MAb) FS17 was raised against the 27-kDa sperm protein. It labeled the sperm body and both flagella by immunofluorescence, though the sperm had to he permeabilized to observe labeling, suggesting that the epitope recognized is not exposed at the cell surface. Addition of FS17 to the KCl extract in the binding assay reduced subsequent binding of egg PMVs. Removal of the 27-kDa protein recognized by FS17 from the sperm extract prevented the binding of egg PMVs in the binding assay and the triggering of the patchy release of polysaccharides when added to eggs. Overall the results suggest that the 27-kDa sperm protein is involved in binding to the egg plasma membrane and can trigger partial activation of the egg .     

Genetic susceptibility to Alzheimer disease

Alzheimer disease (AD) is the leading cause of dementia in the elderly. Less than a decade ago, it was questioned as to whether or not genes were even involved in anything but rare early onset AD. Since that time, using a variety of genetic epidemiological and molecular biological techniques, four loci have been identified that play a role in the genetic susceptibility of AD, AD presents as a prototype of the power of genetic techniques in defining the etiology of a complex disease.     

The application of robotics and mass spectrometry to the characterisation of the Drosophila melanogaster indirect flight muscle proteome

The rapid accumulation of sequence data generated by the various genome sequencingprojects and the generation of expressed sequence tag databases has resulted in the need forthe development of fast and sensitive methods for the identification and characterisation oflarge numbers of gel electrophoretically separated proteins to translate the sequence data intobiological function. To achieve this goal it has been necessary to devise new approaches toprotein analysis: matrix-assisted laser desorption and electrospray mass spectrometry havebecome important protein analytical tools which are both fast and sensitive. When combinedwith a robotic system for the in-gel digestion of electrophoretically separated proteins, itbecomes possible to rapidly identify many proteins by searching databases with MS data. Thepower of this combination of techniques is demonstrated by an analysis of the proteins presentin the myofibrillar lattice of the indirect flight muscle of Drosophila melanogaster. Theproteins were separated by SDS-PAGE and in-gel proteolysis was performed bothautomatically and manually. All 16 major proteins could quickly be identified by massspectrometry. Although most of the protein components were known to be present in theflight muscle, two new components were also identified. The combination of methodsdescribed offers a means for the rapid identification of large numbers of gel separatedproteins.     

Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally

Hybertson, Brooks M., Stuart L. Bursten, Jonathan A. Leff,Young M. Lee, Eric K. Jepson, Chris R. Dewitt, John Zagorski, Hyun G. Cho, and John E. Repine. Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1intratracheally. J. Appl. Physiol.82(1): 226-232, 1997.Interleukin-1 (IL-1) is increased in lunglavages from patients with the acute respiratory distress syndrome, andadministering IL-1 intratracheally causes neutrophil accumulation and aneutrophil-dependent oxidative leak in lungs of rats. In the presentstudy, we found that rats pretreated intraperitoneally with lisofylline[(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (LSF)], an inhibitor of lysophosphatidic acid acyl transferase, which reduces the production of unsaturated phosphatidic acid species,did not develop the lung leak or the related ultrastructural abnormalities that occur after intratracheal administration of IL-1.However, rats pretreated with LSF and then given IL-1 intratracheally did develop the same elevations of lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels and the same increased numbersof lung lavage neutrophils as rats given IL-1 intratracheally. Lungs ofrats given IL-1 intratracheally also had increased unsaturated phosphatidic acid and free acyl (linoleate, linolenate) concentrations compared with untreated rats, and these lipid responses were prevented by pretreatment with LSF. Our results reveal that LSF decreases lungleak and lung lipid alterations without decreasing neutrophil accumulation or lung lavage CINC increases in rats given IL-1 intratracheally.

     

WEEDY ADAPTATION IN SETARIA SPP. I. ISOZYME ANALYSIS OF GENETIC DIVERSITY AND POPULATION GENETIC STRUCTURE IN SETARIA VIRIDIS

Setaria viridis is an important self-pollinating, cosmopolitan weed of temperate regions worldwide. Allozyme markers were used to investigate genetic diversity and structure in 168 accessions (including four S. italica) collected mainly from North America and Eurasia. Genetic diversity in green foxtail, and its population genetic structure, provided important clues about this weed's evolutionary history. Genetic diversity was low, with marked population differentiation: the percentage of polymorphic loci was 25% (0.95 criterion); mean number of alleles per locus was 1.86; mean panmictic heterozygosity was 0.07; and the coefficient of population genetic differentiation was 0.65. A common genotype occurred in 25 accessions distributed in six countries from both the Old World and New World, in a wide variety of ecological situations. Relatively little genetic divergence occurred between Eurasia and North America, with Nei's unbiased genetic identity between the two regions equaling 1.0. Populations from these two continents also had equivalent genetic diversity. Within North America, regional differentiation was indicated by northern and southern groups separated at 43.5° N latitude. No geographic pattern in genetic diversity was found within Eurasia. The size of the geographic range from which populations were sampled was not an accurate indicator of the extent of genetic diversity found among populations from that region. These results suggest that present patterning among green foxtail populations in North America is the consequence of multiple introductions into the New World followed by local adaptation and regional differentiation. Finally, S. italica and several green foxtail varieties did not differ isozymatically from typical forms of green foxtail. This supports the view that S. italica and S. viridis are conspecific, that the former (foxtail millet) is a domesticated form of the latter, and also questions the taxonomic validity of formally recognizing morphological varieties within green foxtail.     

Turing bifurcations with a temporally varying diffusion coefficient

This paper is concerned with the possibility of Turing bifurcations in a reaction-diffusion system in which the diffusion coefficient of one species varies periodically in time. This problem was introduced and investigated numerically by Timm and Okubo (J. Math. Biol. 30, 307, 1992) in the context of predator-prey interactions in plankton populations. Here, I consider the simple case in which the temporal variation in diffusivity has a square-tooth form, alternating between two constant values, with a period that is long compared with the time scale of the kinetics. The analysis is valid for any set of reaction kinetics. I derive explicit expressions for the Floquet multipliers that determine the stability of the steady state, and thereby obtain the conditions for diffusion driven instability to occur. These conditions imply that, depending on the kinetics, the homogeneous equilibrium may be either more or less stable than when the diffusion coefficient is a constant equal to the mean of the variable diffusivity. I go on to consider the form of the solution when diffusion driven instability does occur, and I use perturbation theory to determine the effect of a small temporal variation in the diffusion coefficient on the spatial wavelength of the pattern that results from diffusion driven instability.     

Nonlinear autoregressive analysis of the 3/s ictal electroencephalogram: implications for underlying dynamics

 In a previous study, nonlinear autoregressive (NLAR) models applied to ictal electroencephalogram (EEG) recordings in six patients revealed nonlinear signal interactions that correlated with seizure type and clinical diagnosis. Here we interpret these models from a theoretical viewpoint. Extended models with multiple nonlinear terms are employed to demonstrate the independence of nonlinear dynamical interactions identified in the ‘NLAR fingerprint’ of patients with 3/s seizure discharges. Analysis of the role of periodicity in the EEG signal reveals that the fingerprints reflect the dynamics not only of the periodic discharge itself, but also of the fluctuations of each cycle about an average waveform. A stability analysis is used to make qualitative inferences concerning the network properties of the ictal generators. Finally, the NLAR fingerprint is analyzed in the context of Volterra-Weiner theory. Received: 6 April 1994/Accepted in revised form: 18 November 1994