Glycolipid-peptide conjugate vaccines enhance CD8+ T cell responses against human viral proteins.

An important goal of vaccination against viruses and virus-driven cancers is to elicit cytotoxic CD8+ T cells specific for virus-derived peptides. CD8+ T cell responses can be enhanced by engaging help from natural killer T (NKT) cells. We have produced synthetic vaccines that induce strong peptide-specific CD8+ T cell responses in vivo by incorporating an NKT cell-activating glycolipid. Here we examine the effect of a glycolipid-peptide conjugate vaccine incorporating an NKT cell-activating glycolipid linked to an MHC class I-restricted peptide from a viral antigen in human peripheral blood mononuclear cells. The vaccine induces CD1d-dependent activation of human NKT cells following enzymatic cleavage, activates human dendritic cells in an NKT-cell dependent manner, and generates a pool of activated antigen-specific CD8+ T cells with cytotoxic potential. Compared to unconjugated peptide, the vaccine upregulates expression of genes encoding interferon-γ, CD137 and granzyme B. A similar vaccine incorporating a peptide from the clinically-relevant human papilloma virus (HPV) 16 E7 oncoprotein induces cytotoxicity against peptide-expressing targets in vivo, and elicits a better antitumor response in a model of E7-expressing lung cancer than its unconjugated components. Glycolipid-peptide conjugate vaccines may prove useful for the prevention or treatment of viral infections and tumors that express viral antigens.

Advances in sample preparation in electromigration, chromatographic and mass spectrometric separation methods.

The quality of sample preparation is a key factor in determining the success of analysis. While analysis of pharmaceutically important compounds in biological matrixes has driven forward the development of sample clean-up procedures in last 20 years, today's chemists face an additional challenge: sample preparation and analysis of complex biochemical samples for characterization of genotypic or phenotypic information contained in DNA and proteins. This review focuses on various sample pretreatment methods designed to meet the requirements for the analysis of biopolymers and small drugs in complex matrices. We discuss the advances in development of solid-phase extraction (SPE) sorbents, on-line SPE, membrane-based sample preparation, and sample clean-up of biopolymers prior to their analysis by mass spectrometry.

Judgments of perceptual groups: reliability and sensitivity to stimulus transformation.

The reliability of subjects' judgments of the groups present in dot patterns and the sensitivity of those judgments to stimulus transformation were assessed. The subjects indicated the groups that they saw within random dot patterns, and each judgment was compared with those of other subjects and with their own judgments for related presentations. Within subjects, each pattern appeared in an initial presentation, an identical repetition, and a transformed state (a rotation or a change in scale). Within-subjects judgments were more reliable than between-subjects judgments. An interpretation of within-subjects results was made in relation to predictions made by a formal algorithm of grouping by proximity (the CODE algorithm), which assumes that grouping by proximity is invariant over transformations such as rotations or changes in scale. A slight cost to transforming the patterns was found. The implications for CODE and for using grouping judgments as data are discussed.

Distance and distraction effects in the apprehension of spatial relations.

Theories of the apprehension of spatial relations differ in the predictions they make about the effects of distance between the arguments of spatial relations and the effects of distracting stimuli presented along with the arguments. One theory predicts no effect of distance, another predicts a monotonic increase in reaction time (RT) with distance, and a third predicts a monotonic decrease. Most theories predict slower RTs but reasonable accuracy when distractors are present, but 1 theory predicts chance-level accuracy. These predictions were tested in 3 sentence-picture comparison experiments, in which subjects searched for targets exemplifying the relations above and below. Distance had no effect when no distractors were present. Distractors slowed RT but did not reduce accuracy to chance levels. These results suggest modifications to many of the theories of apprehension.

Use of Sequential Injection technique and robotics for the automation of rhFXIII fluorometric activity assay. case study.

This paper evaluates the feasibility of two systems-the Sequential Injection (SI) analyzer and the Zymark Benchmate (ZB) robot-for the automation of a recombinant human Factor Thirteen (rhFXIII) fluorometric assay. The goal was to develop a routine analytical procedure suitable for the quality control lab environment. The experimental efforts focused on monitoring of the product formation for the condensation reaction between monodansyl cadaverin and dimethyl casein. The acquired kinetic data demonstrated that both systems are capable of automating the solution handling operations associated with the assay. Using a method developed with the SI system, samples containing 0-410 mug/ml of rhFXIII were analyzed, with a throughput of one sample per 8 min, and a total solution consumption of 0.8 ml. The relative standard deviation for 10 injections of 100 mug/ml rhFXIII sample was 0.91%. With the ZB robot, samples containing 0-2500 mug/ml of rhFXIII were analyzed, and the linear response was obtained for a concentration range between 0 and 1250 mug/ml of rhFXIII. The method had a sample throughput of one sample per 11 min and a total solution consumption of 6.3 ml for each analysis. Due to its commercial availability, the ZB system was preferred over the experimental SI system for the purpose of routine automated analysis of a large number of samples in the quality control lab environment.

Evaluating a computational model of perceptual grouping by proximity.

A formal approach to the phenomenon of perceptual grouping by proximity was investigated. Grouping judgments of random dot patterns were made by the CODE algorithm (van Oeffelen & Vos, 1982) and several related algorithms, and these judgments were compared with subjects' grouping judgments for the same stimuli. Each algorithm predicted significantly more subject judgments than would be expected by chance. The more subjects agreed on how a given dot pattern should be grouped, the more successful was the algorithms' ability to match the judgments for that pattern. CODE predicted significantly fewer subject judgments than did some of the other algorithms, largely because of its overemphasis on the extent of interactivity among dots as they are being grouped.

Improved processes for the production and isolation of dynemicin A and large-scale fermentation in a 10,000-liter fermentor.

Supplementing the culture of Micromonospora chersina sp. nov. No. M956-1 with NaI (0.5 mg/l) enhanced the production of dynemicin A by 35-fold in shake flask culture. Homogeneous dynemicin A was obtained from the whole broth extract by Dicalite chromatography, Sephadex LH-20 chromatography and vacuum liquid chromatography. Gram quantities of dynemicin A were obtained from the fermentation of M. chersina sp. nov. No. M956-1 in a 10,000-liter fermentor.

Role of charge suppression and ionic strength in free zone electrophoresis of proteins.

The free zone electrophoretic mobility of proteins can be predicted from the protein's amino acid content by applying a model based on the Debye-Hückle-Henry theory and Henderson-Hasselbalch equation. Calculated mobilities are always greater than actual mobility but a pH-independent proportionality (described by the constant FZ) is found between the two. Thus, determination of a protein's mobility at one pH allows, with the use of the model and FZ, calculation of its mobility at other pH conditions. This leads directly to optimum conditions for the electrophoretic resolution of proteins in capillary zone electrophoresis. The fundamental nature of FZ is examined and found to be a function of a proteins molecular weight, charge, and solution ionic strength. This work aids in explaining the form of previously proposed empirically based equations for peptide and protein mobility.

The transition from algorithm to retrieval in memory-based theories of automaticity.

Two memory-based theories of automaticity were compared. The mixture model and the race model both describe automatization as a transition from algorithmic processing to memory retrieval. The mixture model predicts that, with training, the variability of reaction time will initially increase, and later decrease in a concave downward manner, whereas the race model predicts the variability will decrease only in a concave upward manner. The mixture model predicts that using both algorithm and retrieval on a single trial will be slower than using the algorithm alone, whereas the race model predicts the reverse. The experiments used an alphabet arithmetic task, in which subjects verified equations of the form H + 3 = K and made subjective reports of their strategies on individual trials. Both the variability of reaction times and the pattern of reaction times associated with the strategy reports supported the race model.

Analytical potential of protein A for affinity chromatography of polyclonal and monoclonal antibodies.

Protein A based rapid affinity chromatography for quantitation of various immunoglobulins of class G (IgG) is described. Three-minute analysis using either citrate or phosphate buffers and detection with 220- or 280-nm ultraviolet absorption was found to be optimum for quantitation of IgG from 0.25 to 250 micrograms of IgG on-column with a percent relative standard deviation (% RSD) of 2-3% RSD. The method has a detection limit estimated to be 100 ng of IgG on-column. It has been used to analyze a variety of IgG-containing samples from such diverse sources as hybridoma selection, media cultivation, and purification studies. Gradient elution studies and the relationship of IgG elution to IgG isoelectric point (pI) are also described.

Sedimentation field-flow fractionation: a method for studying particulates in cataractous lens.

It is shown that the technique of sedimentation field-flow fractionation (sedimentation [sed] FFF) can be used to determine the particle content and particle size distribution of normal and cataractous lenses. A 31-year-old normal human lens, for example, showed a particle content of 1.5% by weight with diameters ranging from 0.12 micron to 0.9 micron. The urea insoluble material present in the nuclear and cortical fractions from a densely cataractous lens contained particles ranging from 0.12 micron to 1.7 micron, with average sizes of 0.83 micron and 0.82 micron respectively, for the two fractions. These numbers offer a basis for comparison; their actual values may be shifted slightly either up or down depending on the assessment of particle density. These sizes, which correspond to molecular weights of around 2 X 10(9) dalton, are larger than previously reported for lens particulates. The sed FFF method is thus seen to permit fractionation and size analysis of small amounts of lens material in times less than one hour.