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.     

Emergence of Proteases in Germinating Cucumber Cotyledons and Their Roles in the Two-Step Degradation of Storage Protein

The degradation of storage protein in germinating cucumber seedswas shown to proceed via two distinct steps. First, severalproteases with acidic isoelectric points (pIs) were involvedin solubilization and partial degradation of 11S globulin. Treatmentof seedlings with cycloheximide inhibited this step and theexpression of these proteases. Thus, the first step appearedto be governed by these proteases, which were synthesized denovo after imbibition. The first step was observed in dark-growncotyledons, but the complete degradation of 11S globulin didnot occur in the absence of illumination. An additional protease,with a pI of 4.5, was induced by illumination, and it was involvedin the further cleavage of the partially degraded products ofIIS globulin. Thus, the complete degradation of the storageprotein proceeded via a two-step process in illuminated germinatingseedlings. Light is needed to induce the second step in thedegradation of 11S globulin that supplies the nitrogen requiredfor development of the photosynthetic apparatus in the greeningcotyledon. (Received November 9, 1995; Accepted January 31, 1996)     

Changes in the Chlorophyll Contents of Leaves and in Levels of Cytokinins in Root Exudates during Ripening of Rice Cultivars Nipponbare and Akenohoshi

Changes in fluxes of cytokinins in exudates transported viathe xylem from roots of rice plants cvs. Nipponbare (a standardJapanese cultivar) and Akenohoshi (a slowly senescing cultivar)were measured by mass spectrometry with deuterium-labeled standards.The fluxes of zeatin (Z), trans-ribosylzeatin (trans-RZ), N⁶-isopentenyladenine(iP), and "conjugated Z" (Z in the hydrolysates of highly polarfractions) decreased from heading to the late ripening stagein both cultivars. At the late ripening stage, iP and Z couldno longer be detected, while the flux of N⁶-isopentenyladenosine(iPA) increased slightly. In Akenohoshi, conjugated Z was thepredominant cytokinin from heading to the middle of the ripeningstage. The flux of each of the cytokinins in Akenohoshi washigher than that in Nipponbare at every time point, with theexception of the flux of iPA just after heading. The total concentrationof cytokinins in the xylem exudate of Akenohoshi was higherthan that of Nipponbare after the middle of ripening stage.The chlorophyll content of the third leaves, which were senescingrapidly, was significantly correlated with the flux of totalcytokinins per plant or per unit leaf area. These results suggestthat the larger amounts of cytokinins, in particular conjugatedZ, transported from the roots to the shoots caused the slowsenescence of leaves in Akenohoshi during the ripening stage. (Received May 9, 1994; Accepted July 1, 1995)     

Preparation and antitumor activity of nontoxic lipid A

Summary Highly refined, disaggregated endotoxic glycolipids (B5) from heptose-less (Re) mutant Salmonella typhimurium quantitatively converted to nontoxic (lethality for chick embryos) and nonpyrogenic (fever in rabbits) lipid A by treatment with boiling 0.1 N HCl (B5-HC1). Nontoxic B5-HCl, like toxic B5, caused regression of line-10 tumors and elimination of lymph node metastasis in 27 of 32 (84%) syngeneic strain 2 guinea pigs at a dosage of 150 g. At this dosage, toxic B5 led to a cure in 54 of 67 (81%) tumor-bearing animals. All cured animals rejected a second line-10 tumor cell transplant. This activity depended on combining the toxic or nontoxic endotoxins with mycobacterial trehalose mycolate (P3) and an essentially nontoxic peptide-containing side-fraction (ACP) recovered during the isolation of B5. In contrast to toxic B5 or endotoxins in general, nontoxic B5-HCl did not cause endotoxic shock when combined with adjuvant dipeptide (MDP) and injected IV into guinea pigs. Chemical analysis showed that the phosphate content of nontoxic B5-HCl was about one-half that observed in toxic B5 or in toxic KDO-free lipid A, which was obtained by treating toxic B5 with sodium acetate at pH 4.5 at 100° C (B5-pH 4.5). The molar ratio of glucosamine: phosphorus: fatty acids was 2:1:4 for nontoxic B5-HCl and was 2:2:4 for toxic B5-pH 4.5. These results demonstrate that endotoxic extracts could be selectively detoxified while retaining antitumor properties. Thus, nontoxic B5-HCl may be a potential candidate for immunotherapy of human cancer.Presented at the 72nd Annual Meeting of the American Association for Cancer Research, 1981, and abstract no. 1123 published in the Proceedings of the American Association for Cancer Research, Vol. 22, 1981 Abbreviations used in this paper: ACP, a nontoxic acetone-chloroform precipitated side-fraction of endotoxin that contains (an) ingredient(s) necessary for tumor regression of line-10 tumors in strain 2 guinea pigs; ReGl, endotoxic glycolipids from Re mutant gram-negative bacteria; ReGl-PCP, ReGl extracted with phenol-chloroform-petroleum ether (PCP); B5, refined endotoxin, free of phospholipids, divalent cations and disaggregated; B5-HCl, nontoxic lipid A prepared from B5 by treatment with hydrochloric acid; B5-pH 4.5, toxic lipid A prepared from B5 by treatment with sodium acetate at pH 4.5; lipid A, hydrochloric acid or sodium acetate hydrolysate of ReGl-PCP or B5; MDP, N-acetyl-muramyl-l-seryl-d-isoglutamine; KDO, keto-3-deoxyoctonate     

A Metabolic Study of Huntington’s Disease

Background

Huntington’s disease patients have a number of peripheral manifestations suggestive of metabolic and endocrine abnormalities. We, therefore, investigated a number of metabolic factors in a 24-hour study of Huntington’s disease gene carriers (premanifest and moderate stage II/III) and controls.

Methods

Control (n = 15), premanifest (n = 14) and stage II/III (n = 13) participants were studied with blood sampling over a 24-hour period. A battery of clinical tests including neurological rating and function scales were performed. Visceral and subcutaneous adipose distribution was measured using magnetic resonance imaging. We quantified fasting baseline concentrations of glucose, insulin, cholesterol, triglycerides, lipoprotein (a), fatty acids, amino acids, lactate and osteokines. Leptin and ghrelin were quantified in fasting samples and after a standardised meal. We assessed glucose, insulin, growth hormone and cortisol concentrations during a prolonged oral glucose tolerance test.

Results

We found no highly significant differences in carbohydrate, protein or lipid metabolism markers between healthy controls, premanifest and stage II/III Huntington’s disease subjects. For some markers (osteoprotegerin, tyrosine, lysine, phenylalanine and arginine) there is a suggestion (p values between 0.02 and 0.05) that levels are higher in patients with premanifest HD, but not moderate HD. However, given the large number of statistical tests performed interpretation of these findings must be cautious.

Conclusions

Contrary to previous studies that showed altered levels of metabolic markers in patients with Huntington’s disease, our study did not demonstrate convincing evidence of abnormalities in any of the markers examined. Our analyses were restricted to Huntington’s disease patients not taking neuroleptics, anti-depressants or other medication affecting metabolic pathways. Even with the modest sample sizes studied, the lack of highly significant results, despite many being tested, suggests that the majority of these markers do not differ markedly by disease status.     

Brain Swelling and Loss of Gray and White Matter Differentiation in Human Postmortem Cases by Computed Tomography

The purpose of this study was to evaluate the brain by postmortem computed tomography (PMCT) versus antemortem computed tomography (AMCT) using brains from the same patients. We studied 36 nontraumatic subjects who underwent AMCT, PMCT, and pathological autopsy in our hospital between April 2009 and December 2013. PMCT was performed within 20 h after death, followed by pathological autopsy including the brain. Autopsy confirmed the absence of intracranial disorders that might be related to the cause of death or might affect measurements in our study. Width of the third ventricle, width of the central sulcus, and attenuation in gray matter (GM) and white matter (WM) from the same area of the basal ganglia, centrum semiovale, and high convexity were statistically compared between AMCT and PMCT. Both the width of the third ventricle and the central sulcus were significantly shorter in PMCT than in AMCT (P < 0.0001). GM attenuation increased after death at the level of the centrum semiovale and high convexity, but the differences were not statistically significant considering the differences in attenuation among the different computed tomography scanners. WM attenuation significantly increased after death at all levels (P<0.0001). The differences were larger than the differences in scanners. GM/WM ratio of attenuation was significantly lower by PMCT than by AMCT at all levels (P<0.0001). PMCT showed an increase in WM attenuation, loss of GM–WM differentiation, and brain swelling, evidenced by a decrease in the size of ventricles and sulci.     

Enzymatic synthesis of 2-O-α-d-mannopyranosyl-methyl-α-d-mannopyranoside by a cell-free particulate system of mycobacterium smegmatis

A cell-free particulate enzyme preparation of Mycobacterium smegmatis ATCC 607 catalyzed the transfer of labeled mannose from GDP[¹⁴C]mannose to methyl-α-mannopyranoside (an exogenously added acceptor) to form a product that was characterized to be $\text{2-O-α-}\text{d}\text{-[}{}^{14}\text{C}\text{]}\text{mannopyranosyl-methyl}\text{-α-}\text{D-mannopyranoside}$. This tranmannosylase activity was specific for both the sugar nucleotide donor and methyl monosaccharide acceptor. The reaction was stimulated by the addition of various metal ions and had a pH optimum of 6.0. The apparent K_m of this transmannosylase reaction for methyl-α-d-mannopyranoside was 35 mM.The possible relationship between this “artificial” mannosyl-transfer system and the “natural” system which leads to the formation of the oligomannosides and glycoproteins is discussed.     

Possible association of GroES and antigen 85 proteins with heat resistance of Mycobacterium paratuberculosis

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65 degrees C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65 degrees C (D(65 degrees C) values; times required to reduce the concentration of bacteria by a factor of 10 at 65 degrees C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D(65 degrees C) value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.     

Expression of asparagine synthetase in rice (Oryza sativa) roots in response to nitrogen

The expression of asparagine synthetase (AS; EC 6.3.5.4) in response to externally supplied nitrogen was investigated with respect to enzyme activity and protein levels as detected immunologically in rice ( Oryza sativa ) seedlings. The asparagine content was very low in leaves and roots of nitrogen-starved rice plants but increased significantly after the supply of 1 m M NH₄⁺ to the nutrient solution. While neither AS activity nor AS protein could be detected in leaves and roots prior to the supply of nitrogen, levels became detectable in roots but not in leaves within 12 h of the supply of 1 m M NH₄⁺ or 10 m M glutamine. Other nitrogen compounds, such as nitrate, glutamate, aspartate and asparagine had no effect. Methionine sulfoximine completely inhibited the NH₄⁺-induced accumulation of AS protein but did not affect the glutamine-induced accumulation of the enzyme. The results suggested that glutamine or glutamine-derived metabolites regulate AS expression in rice roots.