A functional annotation of subproteomes in human plasma

The data collected by Human Proteome Organization's Plasma Proteome Pilot project phase was analyzed by members of our working group. Accordingly, a functional annotation of the human plasma proteome was carried out. Here, we report the findings of our analyses. First, bioinformatic analyses were undertaken to determine the likely sources of plasma proteins and to develop a protein interaction network of proteins identified in this project. Second, annotation of these proteins was performed in the context of functional subproteomes involved in the coagulation pathway, the mononuclear phagocytic system, the inflammation pathway, the cardiovascular system, and the liver; as well as the subset of proteins associated with DNA binding activities. Our analyses contributed to the Plasma Proteome Database (http://www.plasmaproteomedatabase.org), an annotated database of plasma proteins identified by HPPP as well as from other published studies. In addition, we address several methodological considerations including the selective enrichment of post-translationally modified proteins by the use of multi-lectin chromatography as well as the use of peptidomic techniques to characterize the low molecular weight proteins in plasma. Furthermore, we have performed additional analyses of peptide identification data to annotate cleavage of signal peptides, sites of intra-membrane proteolysis and post-translational modifications. The HPPP-organized, multi-laboratory effort, as described herein, resulted in much synergy and was essential to the success of this project.     

Quick and reversible inhibition of soybean root nodule growth by nitrate involves a decrease in sucrose supply to nodules

The upper part of a nodulated soybean root hydroponically cultured in a glass bottle was monitored using a computer microscope under controlled environmental conditions, and the diameter of individual nodules was measured from 10-24 d after planting. The diameter of a root nodule attached to the primary root increased from 1 mm to 6 mm for 2 weeks under nitrogen-free conditions. The increase in diameter of the nodules was almost completely stopped after 1 d of supplying 5 mM nitrate, and was due to the cessation of nodule cell expansion. However, nodule growth quickly returned to the normal growth rate following withdrawal of nitrate from the solution. The reversible depression of nodule growth by nitrate was similar to the restriction of photoassimilate supply by continuous dark treatment for 2 d followed by normal light/dark conditions. In addition, the inhibitory effect of nitrate was partially alleviated by the addition of 3% (w/v) sucrose to the medium. Plant leaves were exposed to (11)C or (14)C-labelled carbon dioxide to investigate the effects of 5 mM nitrate on the translocation and distribution of photosynthates to nodules and roots. Supplying 5 mM nitrate stimulated the translocation rate and the distribution of labelled C in nitrate-fed parts of the roots. However, the (14)C partitioning to nodules decreased from 9% to 4% of total (14)C under conditions of 5 mM nitrate supply. These results indicate that the decrease in photoassimilate supply to nodules may be involved in the quick and reversible nitrate inhibition of soybean nodule growth.     

Structural and biochemical dissection of photorespiration in hybrids differing in genome constitution between Diplotaxis tenuifolia (C3-C4) and radish (C3)

We compared the structural, biochemical, and physiological characteristics involved in photorespiration of intergeneric hybrids differing in genome constitution (DtDtR, DtDtRR, and DtRR) between the C(3)-C(4) intermediate species Diplotaxis tenuifolia (DtDt) and the C(3) species radish (Raphanus sativus; RR). The bundle sheath (BS) cells in D. tenuifolia included many centripetally located chloroplasts and mitochondria, but those of radish had only a few chloroplasts and mitochondria. In the hybrids, the numbers of chloroplasts and mitochondria, the ratio of centripetally located organelles to total organelles, and the mitochondrial size in the BS cells increased with an increase in the constitution ratio of the Dt:R genome. The P-protein of glycine decarboxylase (GDC) was confined to the BS mitochondria in D. tenuifolia, whereas in radish, it accumulated more densely in the mesophyll than in the BS mitochondria. In the hybrids, more intense accumulation of GDC in the BS relative to the mesophyll mitochondria occurred with an increase in the Dt:R ratio. These structural and biochemical features in the hybrids were reflected in the gas exchange characteristics of leaves, such as the CO(2) compensation point. Our data indicate that the leaf structure, the intercellular pattern of GDC expression, and the gas exchange characteristics of C(3)-C(4) intermediate photosynthesis are inherited in the hybrids depending on the constitution ratio of the parent genomes. Our findings also demonstrate that the apparent reduced photorespiration in C(3)-C(4) intermediate plants is mainly due to the structural differentiation of mitochondria and chloroplasts in the BS cells combined with the BS-dominant expression of GDC.     

Piezoelectric reciprocal relationship of the membrane motor in the cochlear outer hair cell

It has been shown that the membrane motor in the outer hair cell is driven by the membrane potential. Here we examine whether the motility satisfies the reciprocal relationship, the characteristic of piezoelectricity, by measuring charge displacement induced by stretching the cell with known force. The efficiency of inducing charge displacement was membrane potential dependent. The maximum efficiency of inducing charge displacement by force was approximately 20 fC/nN for 50-microm-long lateral membrane. The efficiency per cell stretching was 0.1 pC/microm. We found that these values are consistent with the reciprocal relationship based on the voltage sensitivity of approximately 20 nm/mV for 50-microm-long cell and force production of 0.1 nN/mV by the cell. We can thus conclude that the membrane motor in the outer hair cell satisfies a necessary condition for piezoelectricity and that the hair cell's piezoelectric coefficient of 20 fC/nN is four orders of magnitude greater than the best man-made material.     

A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonists

In the new high-throughput screening (HTS) campaign, receptor functional assays, 3',5'-cyclic adenosine monophosphate (cAMP), intracellular [Ca(2)+](i), phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays. FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP. A nonradioactive homogeneous HTS assay using HitHunter trade mark enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Galpha(s)-coupled receptor. In the EFC-cAMP assay, the beta-galactosidase (beta-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the beta-gal enzyme acceptor (EA) fragment to form an active beta-gal enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme. Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate. Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample. Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Galpha(s) to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC(50) of 0.3 nM). GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC(50)~0.3 nM) at different cell numbers. The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues. The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway. The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 microM in suspension cells. The assay is very robust, with a Z' value of 0.7 to 0.8. The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies. The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS. The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation. An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.     

High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping

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Diphosphoryl lipid A derived from the lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 is a potent competitive LPS inhibitor in murine macrophage-like J774.1 cells

Abstract Pentaacyl diphosphoryllipid A derived from the nontoxic lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 (RsDPLA) did not induce tumour necrosis factor-α nor interleukin-6 release in the murine macrophage-like cell line J774.1. However, it effectively inhibited the induction of these two cytokines by LPS of Salmonella minnesota Re mutant R595 (ReLPA) in a concentration-dependent manner. Maximal inhibition and half-maximal inhibition occured when the ReLPS to RsDPLA mass ratio was 1:30 and 1:1, respectively. A binding study was performed in the presence of serum to determine whether RsDPLA is competing with ReLPS for LPS binding sites on J774.1 cells. This assay allows the determination of LPS binding to J774.1 cells via a mechanism involving CD14, a receptor for complexes of LPS with LPS binding protein (LBP), and its possible inhibition. The results show that RsDPLA strongly inhibits the binding of ¹²⁵I-labelled ReLPS to J774.1 cells. Maximal and one-half maximal inhibition of binding occured when the ReLPS to RsDPLA mass ratios were 1:2.5 and 1:0.5, respectively. It was found that the inhibition of binding by RsDPLA was much stronger than that by unlabelled ReLPS. These results suggest that RsDPLA is competing with ReLPS for CD14-dependent recognition of LPS on J774.1 cells.     

Stomatal Responses to Water Deficits and Abscisic Acid in Leaves of Sunflower Plants (Helianthus annuus L.) Grown under Different Conditions

The decrease in diffusive conductance of a leaf exposed to waterstress or to exogenous abscisic acid (ABA) was smaller in leavesof sunflower plants (Helianthus annuus L. cv. NK285) that hadbeen grown in a phytotron in humid air than in leaves of sunflowersgrown outdoors. Stomata of the phytotron-grown plants were slowerto close after detachment of a leaf than those of the outdoorplants. When stomata closed rapidly, as they did in detachedleaves and after treatment with ABA, the extent of closure wasvaried over the leaf's surface, in particular in the case ofphytotron-grown plants, and the extent of the heterogeneitywas greater in the phytotrongrown plants than in the outdoorplants. When stomata closed gradually, for example, under conditionsof limited moisture in the soil, closure occurred uniformlyover leaves of plants of both types. The smaller decrease indiffusive conductance of leaves from phytotron-grown plantsafter treatment with ABA resulted from the presence of patcheson the surface in which stomata remained open. The smaller decreaseof diffusive conductance in the phytotron-grown plants underconditions of limited moisture in the soil resulted from theuniformly lower responsiveness of stomata on a leaf to the decreasein water potential. When estimates are made of the intercellularconcentration of CO₂ (Ci) from gas-exchange measurements, heterogeneityin stomatal closure should be monitored when stomata close rapidly,in particular in plants grown in humid air, because heterogeneousstomatal closure can lead to overestimates of Ci. (Received April 18, 1994; Accepted May 25, 1995)     

Oligomannoside biosynthesis in Mycobacterium smegmatis

A cell-free particulate enzyme system of Mycobacterium smegmatis ATCC 607 was shown to catalyze the incorporation of labeled mannose from GDP-[¹⁴C]mannose into endogenous acceptors to form a series of labeled neutral oligomannosides. These oligomannosides were devoid of amino sugar. The major oligomannoside product was characterized to be a trimannoside, O-α-d-mannopyranosyl-(1 → 2)-O-α-d-mannopyranosyl(1 → 2)-d-mannose and represented 46% of the total labeled oligomannoside product. The higher oligomannosides were shown to have either/or both α(1 → 2) and α(1 → 6) glycosidic linkages. A series of unlabeled endogenous oligosaccharides was isolated from the 105,000g supernatant fractions of the cell-free extracts of M. smegmatis and found to be chromatographically similar to the labeled oligomannosides synthesized by the cell-free system. The nature of the endogenous acceptor was not determined.