Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database

HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anti-coagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics.med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay-based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.     

Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.     

The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism

Abelson murine leukemia virus-transformed precursor B lymphocytes from scid (severe combined immunodeficient) mice, like A-MuLV transformants from normal mice, actively rearrange segments of their Ig heavy chain variable region gene locus during growth in culture. Targeting of recombination to appropriate segments appears normal in these lines as evidenced by initial rearrangement of sequences from within the D and JH locus to form aberrant "DJH" rearrangements and secondary rearrangement of sequences from within the VH locus to the aberrant "DJH" intermediates. A detailed analysis of the joints in these rearrangements indicates that the VDJ recombinase in scid pre-B cells can correctly recognize heptamernonamer signal sequences and perform precise endonucleolytic scissions at these sequences. We propose that the scid defect involves the inability of scid precursor lymphocytes to join correctly the cleaved ends of the coding strands of variable region gene segments.     

Polymorphism in NOD2, Crohn's disease, and susceptibility to pulmonary tuberculosis

The nucleotide oligomerization binding domain 2 gene (NOD2) encodes an intracellular receptor for bacterial components, which is expressed in monocytes and is associated with Crohn's Disease (CD). This finding, along with epidemiological evidence, supports a role for infection in the pathogenesis of CD. Speculation that mycobacteria are involved in CD led us to investigate NOD2 in susceptibility to tuberculosis (TB), a global public health problem caused by Mycobacterium tuberculosis. CD-associated NOD2 variants were absent in a case-control study of 640 Gambians, where CD is rare. Novel NOD2 promoter polymorphisms were identified but showed no association with TB in this African population sample.     

On the sufficiency of the velocity field for perception of heading

All models of self-motion from optical flow assume the instantaneous velocity field as input. We tested this assumption for human observers using random-dot displays that simulated translational and circular paths of movement by manipulating the lifetime and displacement of individual dots. For translational movement, observers were equally accurate in judging direction of heading from a velocity field with a two-frame dot life and a direction field in which the magnitudes of displacement were randomized while the radial pattern of directions was preserved, but at chance with a speed field in which the directions were randomized, preserving only magnitude. Accuracy declined with increasing noise in vector directions, but remained below 2.6° with a 90° noise envelope. Thus, the visual system uses the radial morphology of vector directions to determine translational heading and can tolerate large amounts of noise in this pattern. For circular movement, observers were equally accurate with a 2-frame velocity field, 3-frame acceleration displays, and 2-frame and 3-frame direction fields, consistent with the use of the pattern of vector directions to locate the center of rotation. The results indicate that successive independent velocity fields are sufficient for perception of translational and circular heading.     

Homo medicus: The transition to meat eating increased pathogen pressure and the use of pharmacological plants in Homo

The human lineage transitioned to a more carnivorous niche 2.6 mya and evolved a large body size and slower life history, which likely increased zoonotic pathogen pressure. Evidence for this increase includes increased zoonotic infections in modern hunter-gatherers and bushmeat hunters, exceptionally low stomach pH compared to other primates, and divergence in immune-related genes. These all point to change, and probably intensification, in the infectious disease environment of Homo compared to earlier hominins and other apes. At the same time, the brain, an organ in which immune responses are constrained, began to triple in size. We propose that the combination of increased zoonotic pathogen pressure and the challenges of defending a large brain and body from pathogens in a long-lived mammal, selected for intensification of the plant-based self-medication strategies already in place in apes and other primates. In support, there is evidence of medicinal plant use by hominins in the middle Paleolithic, and all cultures today have sophisticated, plant-based medical systems, add spices to food, and regularly consume psychoactive plant substances that are harmful to helminths and other pathogens. We propose that the computational challenges of discovering effective plant-based treatments, the consequent ability to consume more energy-rich animal foods, and the reduced reliance on energetically-costly immune responses helped select for increased cognitive abilities and unique exchange relationships in Homo. In the story of human evolution, which has long emphasized hunting skills, medical skills had an equal role to play.     

The discounting-by-interruptions hypothesis: model and experiment

Experimental animals often prefer small but immediate rewardseven when larger-delayed rewards provide a higher rate of intake.This impulsivity has important implications for models of foragingand cooperation. Behavioral ecologists have hypothesized thatanimals discount delayed rewards because delay imposes a collectionrisk. According to this long-standing hypothesis, delay reducesvalue because an interruption that occurs while an animal iswaiting may prevent it from collecting the delayed reward. Althoughthere have been many experimental demonstrations of animal preferencesfor immediacy, none have included any interruptions. This paperdevelops a simple model of discounting by interruptions andthen tests this model experimentally. The model considers theeffects of interruption rate and duration on choice behavior.The experiment tests the effects of interruptions on the choicebehavior of captive blue jays (Cyanocitta cristata) using afactorial design that manipulates the rate and duration of interruptions.The results do not support the discounting-by-interruptionshypothesis. This represents one of several lines of evidencesuggesting that investigators should seek alternative explanationsof the animal impulsivity.     

Metal cation uptake by yeast: a review

This review addresses metal uptake specifically by yeast. Metal uptake may be passive, active or both, depending on the viability of the biomass, and is influenced by a number of environmental and experimental factors. Uptake is typically accompanied by a degree of ion exchange and, under certain conditions, may be enhanced by the addition of an energy source, Intracellularly accumulated metal is most readily associated with the cell wall and vacuole but may also be bound by other cellular organelles and biomolecules. The intrinsic biochemical, structural and genetic properties of the yeast cell along with environmental conditions are crucial for its survival when exposed to toxic metals. Conditions of pH, temperature and the presence of additional ions, amongst others, have varying effects on the metal uptake process. We conclude that yeasts have contributed significantly to our understanding of the metal uptake process and suggest directions for future work.     

Shapes and sizes of badger territories

We examine closely the models, methods and conclusions of Doncaster and Woodroffe (1993; Oikos 66: 88–93 who argued that den or main sett sites of clans of badgers, Meles meles , are particularly important in determining territory shape and size, and hence influence the size of social group. We consider a realistic alternative hypothesis which allows the key assertion by Doncaster and Woodroffe to be directly tested. We show that a Dirichlet tessellation model that does not give a major role to the main setts fits data from several studies – two of those considered by Doncaster and Woodroffe, and a more recent and extensive one – significantly better than Doncaster and Woodroffe's model. For the majority of territories, especially in the most extensive data set, differences in territory shape and size under the two models are substantial, suggesting that a different biological mechanism is at work, as well as or instead of dependence on main sett locations.     

Photorespiratory mutants of the mitochondrial conversion of glycine to serine

Two mutants of barley (Hordeum vulgare L.), LaPr 85/55 and LaPr 87/30, have been isolated that accumulate glycine, with a concomitant reduction in the aminodonors glutamate and alanine, when transferred to air. Studies have shown that these plants have wild-type levels of serine transhydroxymethylase (EC 2.1.2.1) activity. When supplied ¹⁴CO₂, 48 and 66% of the supplied carbon was retained as glycine in LaPr 85/55 and LaPr 87/30, respectively, compared with a value of 11% for the wild type. In the short-term, both mutant plants are unable to metabolize [¹⁴C] glycine, but when fed the isotope for 2 hours, LaPr 85/55 was able to metabolize most (70%) of the supplied carbon into sugars with only 15% remaining in glycine. LaPr 87/30, however, was unable to metabolize more than 4% of the supplied carbon into sugars even after 2 hours. Measurement of glycine decarboxylase (EC 2.1.2.10) activity via the glycine-bicarbonate exchange reaction showed LaPr 85/55 to have approximately 70% wild-type activity with LaPr 87/30 having only 14% wild-type activity. The approximation of LaPr 85/55 to wild-type activities was maintained for ¹⁴CO₂ release from [¹⁴C]glycine feeding and ammonia accumulation in the presence of methionine sulphoximine with the equivalent rates for LaPr 87/30 being less than 40% and 10%, respectively. CO₂ fixation rates for the mutants fell to between 35 and 40% of wild-type rates within 10 min of transfer to air. This was shown to be partly due to a run down of aminodonors, because when 40 millimolar serine was supplied through the xylem stream these rates recovered for both mutants to 70% of the wild-type rate. These data suggested a mutation in a glycine transport system for LaPr 85/55 and in the proteins of glycine decarboxylase for LaPr 87/30. Western blotting with antisera to the P, H, T, and L proteins of glycine decarboxylase showed cross-reaction against all four proteins for LaPr 85/55 but little cross-reaction against P or H protein for LaPr 87/30, reaffirming the possibility of a transport mutation in LaPr 85/55. We also suggest that genes for P and H proteins could be either coordinately regulated or that one protein is undetectable or unstable in the absence of the other.