Peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display

The human Plasma Proteome Project pilot phase aims to analyze serum and plasma specimens to elucidate specimen characteristics by various proteomic techniques to ensure sufficient sample quality for the HUPO main phase. We used our proprietary peptidomics technologies to analyze the samples distributed by HUPO. Peptidomics summarizes technologies for visualization, quantitation, and identification of the low-molecular-weight proteome (<15 kDa), the "peptidome." We analyzed all four HUPO specimens (EDTA plasma, citrate plasma, heparin plasma, and serum) from African- and Asian-American donors and compared them to in-house collected Caucasian specimens. One main finding focuses on the most suitable method of plasma specimen collection. Gentle platelet removal from plasma samples is beneficial for improved specificity. Platelet contamination or activation of platelets by low temperature prior to their removal leads to distinct and multiple peptide signals in plasma samples. Two different specimen collection protocols for platelet-poor plasma are recommended. Further emphasis is placed on the differences between plasma and serum on a peptidomic level. A large number of peptides, many of them in rather high abundance, are only present in serum and not detectable in plasma. This ex vivo generation of multiple peptides hampers discovery efforts and is caused by a variety of factors: the release of platelet-derived peptides, other peptides derived from cellular components or the clot, enzymatic activities of coagulation cascades, and other proteases. We conclude that specimen collection is a crucial step for successful peptide biomarker discovery in human blood samples. For analysis of the low-molecular-weight proteome, we recommend the use of platelet-depleted EDTA or citrate plasma.     

A protecting group for carboxylic acids that can be photolyzed by visible light

We report on a photolabile protecting (caging) group that is new for carboxylic acids. Unlike previously used caging groups for carboxylic acids, it can be photolyzed rapidly and efficiently in the visible wavelength region. The caging group 7-N,N-diethyl aminocoumarin (DECM) was used to cage the gamma-carboxyl group of glutamic acid, which is also a neurotransmitter. The caged compound has a major absorption band with a maximum at 390 nm (epsilon(390) = 13651 M(-)(1) cm(-)(1)). Experiments are performed at 400 nm (epsilon(400) = 12232 M(-)(1) cm(-)(1)) and longer wavelengths. DECM-caged glutamate is water soluble and stable at pH 7.4 and 22 degrees C. It photolyzes rapidly in aqueous solution to release glutamic acid within 3 micros with a quantum yield of 0.11 +/- 0.008 in the visible region. In whole-cell current-recording experiments, using HEK-293 cells expressing glutamate receptors and visible light for photolysis, DECM-caged glutamate and its photolytic byproducts were found to be biologically inert. Neurotransmitter receptors that are activated by various carboxyl-group-containing compounds play a central role in signal transmission between approximately 10(12) neurons of the nervous system. Caged neurotransmitters have become an essential tool in transient kinetic investigations of the mechanism of action of neurotransmitter receptors. Previously uncaging the compounds suitable for transient kinetic investigations required ultraviolet light and expensive lasers, and, therefore, special precautions. The availability of caged neurotransmitters suitable for transient kinetic investigations that can be photolyzed by visible light allows the use of simple-to-use, readily available inexpensive light sources, thereby opening up this important field to an increasing number of investigators.     

Picrotoxin inhibition mechanism of a gamma-aminobutyric acid A receptor investigated by a laser-pulse photolysis technique

The gamma-aminobutyric acid(A) (GABA(A)) receptor, a major inhibitory neurotransmitter receptor, belongs to a family of membrane-bound proteins that regulate signal transmission between approximately 10(12) cells of the nervous system. It plays a major role in many neurological disorders, including epilepsy. It is the target of many pharmacological agents, including the convulsant picrotoxin. Here, we present the mechanism of inhibition by picrotoxin of the rat alpha1beta2gamma2L GABA(A) receptor investigated using rapid kinetic techniques in combination with whole-cell current recordings. The following new results were obtained by using transient kinetic techniques, the cell-flow method and the laser-pulse photolysis (LaPP) technique with a microsecond to millisecond time resolution. (i) The apparent dissociation constant of picrotoxin for the open-channel form of the receptor was approximately 5 times higher than that of the closed-channel form. (ii) Picrotoxin increased the channel-closing rate constant (k(cl)) approximately 4-fold, while the rate constant for channel opening (k(op)) remained essentially unaffected. (iii) The mechanism indicates that picrotoxin binds to an allosteric site of the receptor with higher affinity for the closed-channel form than for the open-channel form and thereby inhibits the receptor by decreasing 4-fold its channel-opening equilibrium constant [Phi(I)(-)(1) = k(op(I))/k(cl(I))]. (iv) The mechanism further indicates that compounds that bind with equal affinity to the picrotoxin-binding site on the open-channel form of the receptor and the closed-channel form will not affect the channel-opening equilibrium and can, therefore, displace picrotoxin and prevent inhibition of the GABA(A) receptor by picrotoxin. Such compounds may be therapeutically useful in counteracting the effects of compounds and diseases that unfavorably affect the channel-opening equilibrium of the receptor channel.     

Virus particles in an internal parasite, Portunion conformis (Crustacea: Isopoda: Entoniscidae), and its marine crab host, Hemigrapsus oregonensis

Based on morphological evidence and preliminary physicochemical data, we report the first picornavirus from crustacean hosts. The viral particles are widespread in the tissues of an isopodan parasitic castrator, Portunion conformis, and its shore crab host, Hemigrapsus oregonensis, collected in San Francisco Bay, California. Less frequently, infected cells of the parasitic isopod also contain larger viral particles.     

Maternal nutrient restriction reduces concentrations of amino acids and polyamines in ovine maternal and fetal plasma and fetal fluids

Amino acids and polyamines are essential for placental and fetal growth, but little is known about their availability in the conceptus in response to maternal undernutrition. We hypothesized that maternal nutrient restriction reduces concentrations of amino acids and polyamines in the ovine conceptus. This hypothesis was tested in nutrient-restricted ewes between Days 28 and 78 (experiment 1) and between Days 28 and 135 (experiment 2) of gestation. In both experiments, ewes were assigned randomly on Day 28 of gestation to a control group fed 100% of National Research Council (NRC) nutrient requirements and to an nutrient-restricted group fed 50% of NRC requirements. Every 7 days beginning on Day 28 of gestation, ewes were weighed and rations adjusted for changes in body weight. On Day 78 of gestation, blood samples were obtained from the uterine artery and umbilical vein for analysis. In experiment 2, nutrient-restricted ewes on Day 78 of gestation either continued to be fed 50% of NRC requirements or were realimented to 100% of NRC requirements until Day 135. Fetal weight was reduced in nutrient-restricted ewes at both Day 78 (32%) and Day 135 (15%) compared with controls. Nutritional restriction markedly reduced (P < 0.05) concentrations of total alpha-amino acids (particularly serine, arginine-family amino acids, and branched-chain amino acids) and polyamines in maternal and fetal plasma and in fetal allantoic and amniotic fluids at both mid and late gestation. Realimentation of nutrient-restricted ewes increased (P < 0.05) concentrations of total alpha-amino acids and polyamines in all the measured compartments and prevented intrauterine growth retardation. These novel findings demonstrate that 50% global nutrient restriction decreases concentrations of amino acids and polyamines in the ovine conceptus that could adversely impact key fetal functions. The results have important implications for understanding the mechanisms responsible for both intrauterine growth retardation and developmental origins of adult disease.     

Mechanism-based discovery of small molecules that prevent noncompetitive inhibition by cocaine and MK-801 mediated by two different sites on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) belongs to a group of five structurally related membrane proteins that play a major role in the communication between approximately 10(12) cells of the mammalian nervous system. The receptor is inhibited by both abused drugs and therapeutic agents. During the past two decades, many attempts have been made to find compounds that prevent cocaine inhibition of this protein. The use of newly developed transient kinetic techniques in investigations of the inhibition of the receptor by cocaine and MK-801 led to an inhibition mechanism not previously proposed. It was observed that the receptor contains two inhibitory sites: one that equilibrates with the tested noncompetitive inhibitors within approximately 50 ms, and a second site that equilibrates with inhibitors within approximately 1 s. The mechanism of inhibition of the rapidly equilibrating inhibitory site has been investigated, and based on that mechanism, the first evidence that small organic molecules exist that prevent inhibition of the rapidly equilibrating inhibitory site was obtained. These compounds did not prevent the inhibition due to the slowly equilibrating inhibitory site. Here, we present the first evidence that a compound (3-acetoxy ecgonine methyl ester) exists that prevents inhibition of the slowly equilibrating inhibitory site and that the mechanism of inhibition of this site differs from that of the rapidly equilibrating site. BC3H1 cells containing a fetal mouse muscle-type nAChR were used, and the receptor was activated by carbamoylcholine. The resulting whole-cell current due to the nondesensitized nAChR was determined. Because the nAChR desensitizes rapidly, the measurements required the use of a transient kinetic technique with a time resolution of 10 ms; the cell-flow technique was used. Inhibitors and compounds that alleviate inhibition were tested by determining their effects on the whole-cell current due to activation of the nAChR by carbamoylcholine.     

Genes malh and pagl of Clostridium acetobutylicum ATCC 824 encode NAD+- and Mn2+-dependent phospho-alpha-glucosidase(s)

The genome of Clostridium acetobutylicum 824 contains two genes encoding NAD+, Mn2+, and dithiothreitol-dependent phospho-alpha-glucosidases that can be assigned to family 4 of the glycosylhydrolase superfamily. The two genes, designated malh (maltose 6-phosphate hydrolase) and pagl (phospho-alpha-glucosidase), respectively, reside in separate operons that also encode proteins of the phosphoenolpyruvate-dependent:sugar phosphotransferase system. C. acetobutylicum grows on a variety of alpha-linked glucosides, including maltose, methyl-alpha-d-glucoside, and the five isomers of sucrose. In the presence of the requisite cofactors, extracts of these cells readily hydrolyzed the chromogenic substrate p-nitrophenyl-alpha-d-glucopyranoside 6-phosphate, but whether hydrolysis reflected expression of enzymes encoded by the malh or pagl genes was not discernible by spectrophotometric analysis or polyacrylamide gel electrophoresis. Resolution of this question required the cloning of the malh and pagl genes, and subsequent high expression, purification, and characterization of maltose-6'-phosphate hydrolase (MalH) and phospho-alpha-glucosidase (PagL), respectively. MalH and PagL exhibit 50% residue identity, and in solution are tetramers comprising similar sized ( approximately 50 kDa) subunits. The two proteins cross-react with polyclonal rabbit antibody against phospho-alpha-glucosidase from Fusobacterium mortiferum. Purified MalH and PagL cleaved p-nitrophenyl-alpha-d-glucopyranoside 6-phosphate with comparable efficiency, but only MalH catalyzed the hydrolysis of disaccharide 6'-phosphates formed via the phosphoenolpyruvate-dependent:sugar phosphotransferase system. Importantly, analysis of the proteome of C. acetobutylicum 824 by electrospray ionization-mass spectrometry confirmed expression of MalH during growth on many alpha-glucosides tested. Site-directed changes C169S and D170N yielded full-length, but catalytically inactive MalH. Of the two putative operons, our findings suggest that only proteins encoded by the mal operon participate in the dissimilation of maltose and related O-alpha-linked glucosides by C. acetobutylicum 824.     

FcR interactions do not play a major role in inhibition of experimental autoimmune encephalomyelitis by anti-CD154 monoclonal antibodies

It has been demonstrated that anti-CD154 mAb treatment effectively inhibits the development of experimental autoimmune encephalomyelitis (EAE). However, although it appears to prevent the induction of Th1 cells and reactivation of encephalitogenic T cells within the CNS, little information is available regarding the involvement of alternative mechanisms, nor has the contribution of Fc effector mechanisms in this context been addressed. By contrast, efficacy of anti-CD154 mAbs in models of allotransplantation has been reported to involve long-term unresponsiveness, potentially via activation of T regulatory cells, and recently was reported to depend on Fc-dependent functions, such as activated T cell depletion through FcgammaR or complement. In this study we demonstrate that anti-CD154 mAb treatment inhibits EAE development in SJL mice without apparent long-term unresponsiveness or active suppression of disease. To address whether the mechanism of inhibition of EAE by anti-CD154 mAb depends on its Fc effector interactions, we compared an anti-CD154 mAb with its aglycosyl counterpart with severely impaired FcgammaR binding and reduced complement binding activity with regard to their ability to inhibit clinical signs of EAE and report that both forms of the Ab are similarly protective. This observation was largely confirmed by the extent of leukocyte infiltration of the CNS; however, mice treated with the aglycosyl form may display slightly more proteolipid protein 139-151-specific immune reactivity. It is concluded that FcR interactions do not play a major role in the protective effect of anti-CD154 mAb in the context of EAE, though they may contribute to the full abrogation of peripheral peptide-specific lymphocyte responses.     

Application of immunoproteomics to rapid cytokine detection

Cytokines are pivotal to a balanced innate or cell-mediated immune response, and can be indicative of disease progression and/or resolution. Methods to measure key cytokines rapidly with accuracy, precision, and sensitivity are consequently important. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative, semi-quantitative approach described here, uses an immunological capture step and a mass spectral readout. The goal of the assay is speed rather than sensitivity or precision.     

ImmunoTrack: the novel antibody-based technology for tracing in animal health

This paper describes a novel antibody-based livestock movement control tool and method of meat allocation, both in livestock husbandry as well as during the meat-processing chain. Immuno Track fulfills diverse prerequisites and meets regulatory demands which are substantial for a successful monitoring technology: (i) the induction of long-lasting antibody responses detectable onsite throughout the whole mast period of pigs, (ii) a single immunization injection with protein derivatives is sufficient to evoke a strong epitope-specific antibody response, and (iii) the complete degradation of the protein markers after the antibody response has been triggered in meatproducing animals such as cattle or pigs. There are diverse fields of application for the Immuno-Track marker technology, such as in quality meat programs, as compliance markers for animal vaccines or as a tool for verification of origin. Combination of this monitoring technology with the husbandry and identification databases for cattle and pigs within the European Community will lead to greater transparency in meat production, thereby regaining consumers' trust in concomitant structures of the meat-producing industry.