Properties of blocking and non-blocking monoclonal antibodies specific for human macrophage galactose-type C-type lectin (MGL/ClecSF10A/CD301)

Monoclonal antibodies (mAbs) specific for the human macrophage galactose-type calcium-type lectin (MGL) were established. The recombinant extracellular domain of MGL was used to immunize a mouse, and 10 hybridoma clones were obtained. Binding of recombinant MGL to asialo-bovine submaxillary mucin was shown to be blocked by mAbs MLD-1, 4 and 6. Immunoprecipitation of MGL from lysates of COS-1 cells transfected with MGL cDNA (form 6A) was achieved with mAbs MLD-1, 4, 7, 8 and 16. Chimeric recombinant proteins between human MGL and mouse MGL1 were used to determine the location of the epitopes for these mAbs. mAbs MLD-8, 13, 15 and 16 interacted with the amino terminal side of the conserved WVDGTD sequence immediately upstream of QPD, whereas mAbs MLD-7, 12 and 17 interacted with the other side. mAbs MLD-1, 4, and 6 apparently required both sides of this boundary. mAbs MLD-15 and 16 were shown to recognize the protein products of alternatively spliced mRNA 6A/8A and 6C/8A, having deletions at the boundary of exons 7 and 8, in addition to full length and other spliced forms of MGL (6A, 6B and 6C), whereas the other mAbs bound only full length and forms 6A, 6B and 6C.     

Dual subcellular localization in the endoplasmic reticulum and peroxisomes and a vital role in protecting against oxidative stress of fatty aldehyde dehydrogenase are achieved by alternative splicing

Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner.     

Proposed minimum reporting standards for chemical analysis

There is a general consensus that supports the need for standardized reporting of metadata or information describing large-scale metabolomics and other functional genomics data sets. Reporting of standard metadata provides a biological and empirical context for the data, facilitates experimental replication, and enables the re-interrogation and comparison of data by others. Accordingly, the Metabolomics Standards Initiative is building a general consensus concerning the minimum reporting standards for metabolomics experiments of which the Chemical Analysis Working Group (CAWG) is a member of this community effort. This article proposes the minimum reporting standards related to the chemical analysis aspects of metabolomics experiments including: sample preparation, experimental analysis, quality control, metabolite identification, and data pre-processing. These minimum standards currently focus mostly upon mass spectrometry and nuclear magnetic resonance spectroscopy due to the popularity of these techniques in metabolomics. However, additional input concerning other techniques is welcomed and can be provided via the CAWG on-line discussion forum at or . Further, community input related to this document can also be provided via this electronic forum. The contents of this paper do not necessarily reflect any position of the Government or the opinion of the Food and Drug Administration Sponsor: Metabolomics Society http://www.metabolomicssociety.org/ Reference: http://msi-workgroups.sourceforge.net/bio-metadata/reporting/pbc/ http://msi-workgroups.sourceforge.net/chemical-analysis/ Version: Revision: 5.1 Date: 09 January, 2007     

Effect of electrical stimulation of antagonist muscles for voluntary motor drive

Voluntary motor drive is an important central command that descends via the corticospinal tract to initiate muscle contraction. When electrical stimulation (ES) is applied to an antagonist or agonist muscle, it changes the agonist muscle’s representative motor cortex and thus its voluntary motor drive. In this study, we used a reaction time task to compare the effects of weak and strong ES of the antagonist or agonist muscle during the premotor period of a wrist extension. We recorded motor evoked potentials (MEPs) induced by transcranial magnetic stimulation (TMS) that was applied to the extensor carpi radialis (ECR; agonist) and flexor carpi radialis (FCR; antagonist). When stronger ES intensities were applied to the antagonist, the MEP control ratio in the ECR significantly increased during the premotor time. Furthermore, the MEP control ratio with stronger antagonist ES intensity was significantly larger than that in the agonist for the same ES intensity. In the FCR, the MEP control ratio was also significantly greater at the strong ES intensity than at the weak ES intensity. Furthermore, the MEP control ratio in the antagonist with a strong ES intensity was significantly larger than that in the agonist with the same ES intensity. These results suggest that agonist corticomotor excitability might be enhanced by ES of the antagonist, which in turn strongly activates the descending motor system in the preparation of agonist contraction.     

Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS

Root exudates released into soil have important functions in mobilizing metal micronutrients and for causing selective enrichment of plant beneficial soil micro-organisms that colonize the rhizosphere. Analysis of plant root exudates typically has involved chromatographic methods that rely on a priori knowledge of which compounds might be present. In the research reported here, the combination of multinuclear and 2-D NMR with GC-MS and high-resolution MS provided de novo identification of a number of components directly in crude root exudates of different plant types. This approach was applied to examine the role of exudate metal ion ligands (MIL) in the acquisition of Cd and transition metals by barley and wheat. The exudation of mugineic acids and malate was enhanced by Fe deficiency. which in turn led to an increase in the tissue content of Cu, Mn, and Zn. The presence of elevated Cd maintained at a free activity pCd of 8.8 (10(-8.8) M), resulted in reduced phytosiderophore production by Fe deficient plants. The buffer morpholinoethane sulfonate (MES), which is commonly used in chelator-buffering nutrient solutions, was detected in the root exudate mixture, suggesting uptake and re-secretion of this compound by the roots. The ability to detect this compound in complex mixtures containing organic acids, amino acids, and other substances suggests that the analytical methods used here provide an unbiased method for simultaneous detection of all major components contained in root exudates.     

Modeling associative learning with generalization for a case of warning signals

Animals associative learning plays a crucial role in many intraspecific or interspecific interactions, involving an animals use of information on its interacting counterparts. Here, we present a theoretical model that captures the basic features of an animals associative learning, which may involve generalization, for a simplest case of warning signals. Specifically, we derive formulae for the average level of associative memory as functions of a few parameters that reflect the population density of prey, predators efficiency of prey detection, and the properties of predators associative learning, including generalization and memory decay. This average level of associative memory is of central importance in determining preys fitness and, thus, the evolution of warning signals (i.e. aposematism). The derived formula also shows that another species with similar signal enhances the fitness of an aposematic species of concern as long as their signal is similar enough for generalization to occur. The model developed here can be extended to more complicated cases and the basic idea can be applied to modeling other interactions involving associative learning with generalization.     

Autophosphorylation of p110delta phosphoinositide 3-kinase: a new paradigm for the regulation of lipid kinases in vitro and in vivo

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases which also possess an in vitro protein kinase activity towards themselves or their adaptor proteins. The physiological relevance of these phosphorylations is unclear at present. Here, the protein kinase activity of the tyrosine kinase-linked PI3K, p110delta, is characterized and its functional impact assessed. In vitro autophosphorylation of p110delta completely down-regulates its lipid kinase activity. The single site of autophosphorylation was mapped to Ser1039 at the C-terminus of p110delta. Antisera specific for phospho-Ser1039 revealed a very low level of phosphorylation of this residue in cell lines. However, p110delta that is recruited to activated receptors (such as CD28 in T cells) shows a time-dependent increase in Ser1039 phosphorylation and a concomitant decrease in associated lipid kinase activity. Treatment of cells with okadaic acid, an inhibitor of Ser/Thr phosphatases, also dramatically increases the level of Ser1039-phosphorylated p110delta. LY294002 and wortmannin blocked these in vivo increases in Ser1039 phosphorylation, consistent with the notion that PI3Ks, and possibly p110delta itself, are involved in the in vivo phosphorylation of p110delta. In summary, we show that PI3Ks are subject to regulatory phosphorylations in vivo similar to those identified under in vitro conditions, identifying a new level of control of these signalling molecules.