A solid-phase immunoassay for the binding of cartilage proteoglycan to hyaluronic acid

A solid-phase assay for detecting the binding of cartilage proteoglycan (PG) to hyaluronic acid (HA) is described. In the assay, HA is immobilized on protamine-treated microtiter wells, the wells are incubated with PG monomer and antibody to PG monomer, and then an ELISA system is used to detect binding of the PG to HA. The specificity of the assay is indicated by the failure to detect PG binding to chondroitin sulfate or albumin-coated microtiter wells, the absence of binding with tryptic fragments of PG monomer other than the HA-binding segment, the loss of binding after reduction and alkylation of PG monomer, and the inhibition of binding by preincubation of PG monomer with small amounts of HA. In contrast to the HA-PG interaction in solution, hyaluronidase digestion of HA does not affect its ability to inhibit the reaction of PG monomer with immobilized HA. The microtiter well-based assay appears to be a rapid, simple, and potentially versatile method for studying interactions with HA.     

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronan (hyaluronic acid) in biological fluids

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronic acid (HA) is described. The principle of the method depends on the specific binding of HA to the hyaluronic acid-binding region (HABR) of proteoglycan (PG) monomers. The remaining uncomplexed PG monomers were determined by incubation with specific monoclonal antibodies to HABR followed by addition of polyclonal antibodies against PG monomers and enzyme-conjugated antibodies. The HA in samples was quantified by comparing their inhibitory capacity in the assay against a standard inhibition curve obtained using highly purified HA. This method was used to quantitate HA at nanogram levels in normal sera and synovial fluids. The level in normal human sera was found to be 28 +/- 17 ng/ml which compared favorably with values obtained using a commercial radioassay kit on the same samples. The assay was used to measure HA in synovial fluid from patients with rheumatoid and osteoarthritis and the results obtained were comparable with data published by others.     

Arabidopsis phosphatidylglycerophosphate phosphatase 1 involved in phosphatidylglycerol biosynthesis and photosynthetic function

Phosphatidylglycerol (PG) is an indispensable lipid constituent of photosynthetic membranes, whose function is essential in photosynthetic activity. In higher plants, the biological function of the last step of PG biosynthesis remains elusive because an enzyme catalyzing this reaction step, namely phosphatidylglycerophosphate phosphatase (PGPP), has been a missing piece in the entire glycerolipid metabolic map. Here, we report the identification and characterization of AtPGPP1 encoding a PGPP in Arabidopsis thaliana. Heterologous expression of AtPGPP1 in yeast Δgep4 complemented growth phenotype and PG‐producing activity, suggesting that AtPGPP1 encodes a functional PGPP. The GUS reporter assay showed that AtPGPP1 was preferentially expressed in hypocotyl, vasculatures, trichomes, guard cells, and stigmas. A subcellular localization study with GFP reporter indicated that AtPGPP1 is mainly localized at chloroplasts. A T‐DNA‐tagged knockout mutant of AtPGPP1, designated pgpp1‐1, showed pale green phenotype with reduced PG and chlorophyll contents but no defect in embryo development. In the pgpp1‐1 mutant, ultrastructure of plastids indicated defective development of chloroplasts and measurement of photosynthetic parameters showed impaired photosynthetic activity. These results suggest that AtPGPP1 is a primary plastidic PGPP required for PG biosynthesis and photosynthetic function in Arabidopsis.     

Glycolate and 2-phosphoglycolate content of tissues measured by ion chromatography coupled to mass spectrometry

Glycolate and 2-phosphoglycolate (PG) are 2-carbon monocarboxylic acids with ill-defined metabolic roles. Their concentrations have not yet been described in tissues apart from body fluids and erythrocytes. We describe the use of ion chromatography coupled with mass spectrometry (IC-MS) to quantify levels of glycolate and PG in tissue. Sample preparation and analysis can be performed within an hour. Low concentrations of glycolate (12-48 nmol/g) and PG (4-17 nmol/g) were detected in all tissues. The availability of this IC-MS assay will facilitate investigations of the origin, function, and metabolism of glycolate and PG in tissues.     

6-Phosphogluconate Dehydrogenase Mechanism: EVIDENCE FOR ALLOSTERIC MODULATION BY SUBSTRATE

The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG ^D(V/K) and ^D(V) are 1.68 and 2.46, respectively, whereas the presence of 6PG increases ^D(V/K) to 2.84 and decreases ^D(V) to 1.38. A similar increase of ^T(V/K) is observed as 6PG builds up in the reaction mixture. These data indicate that in the absence of 6PG, a slow step, which precedes the chemical process, is rate-limiting for the reaction, whereas in the presence of 6PG, the rate-limiting step follows the isotope-sensitive step. Kinetic analysis of reductive carboxylation shows that 6PG at low concentrations decreases the K_m of Ru5P, whereas at higher concentrations, the usual competitive pattern is observed. These data indicate that full activity of 6PGDH is achieved when one subunit carries out the catalysis and the other subunit carries an unreacted 6PG. Thus, 6PG is like an allosteric activator of 6PGDH.     

A sensitive GLC-method for component sugars andO-glycosidic linkage monosaccharides of cartilage proteoglycans

A method is described for the measurement ofN-acetylgalactosamine,N-acetylglucosamine, galactose, mannose and xylose present in the different carbohydrate chains of cartilage proteoglycans (PG). Bovine articular cartilage PG samples corresponding to the minimum of 1 nmol of each monosaccharide were reproducibly quantified following hydrolysis with 2 M HCl and derivatization into alditol acetates. An on-column injection mode and an OV-1701 fused silica capillary column were used for chromatography.Alkaline borohydride treatment of the PG was exploited to reduce the acid labile xylose in the base of the chondroitin sulphate chain into more stable xylitol, allowing the assay of chondroitin sulfate chain length as anN-acetylgalactosamine/xylose ratio. A novel procedure is described for the measurement of the galactosaminitol evolving from the protein linkage of oligosaccharides and of keratan sulphate.     

Effects of the proapoptotic drug prodigiosin on cell cycle-related proteins in Jurkat T cells

Prodigiosin (PG) is a red pigment produced by Serratia marcescens with immunosuppressive and apoptotic activities. In this study, we sought to examine the effect of PG on cell cycle-related proteins. The antiproliferative activity of PG was tested using human Jurkat leukaemia T cells in culture. PG-inhibited cell proliferation was determined using thymidine incorporation assay. PG-arrested cell cycle was analysed using immunoblot analysis with specific antibodies against cell cycle-related proteins and kinase assays of cdk2. Apoptosis was determined by Hoechst staining and analysis of DNA fragmentation. PG inhibited cyclin E, cdk2, p27 and p21, the induction of the cyclin A-cdk2 and cyclin E-cdk2 kinase activity, and the phosphorylation of Rb in leukaemic Jurkat cells. We confirmed that PG induces apoptosis by the characteristic DNA laddering pattern and condensed nuclei or apoptotic bodies identified by fluorescence microscopy. These results indicate that PG and other family members form a new group of molecules with a common mechanism of action and specific molecular targets, raising the possibility of their therapeutic use as antineoplastic drugs.     

Thymic hormones and prostaglandins. II. Synergistic effect on mouse spontaneous rosette forming cells

Prostaglandins (PGs) have been assumed to play a role in the biological activity of thymic hormones (TH). Indeed, it has been shown that type E-PGs are able to mimic the action of several TH. Moreover, indomethacin interferes in the rosette assay, which still represents the most commonly used bioassay for the evaluation of TH and, in particular thymulin levels, in biological fluids. Previously, our attempt to modulate PG production by different TH showed that none of the TH tested affect PGE2, 6-keto-PGF1 alpha, PGF2 alpha and TXB2 production by spleen cells from control and thymectomized (Tx) mice, while indomethacin was able to inhibit the spontaneous PG production. Here, we investigated a possible role for each endogenously produced PG in the experimental conditions of the rosette assay, in order to define: 1) whether or not there was a specificity of action of a given PG; and 2) to analyze the pattern of action between thymulin and the endogenously produced PGs. We demonstrated that PGE2 and 6-keto-PGF1 alpha are the PGs which are physiologically involved in the rosette assay, according to their levels of endogenous production, and that they are able to synergize with thymulin. This synergy was demonstrated in two ways: 1) by adding anti-PGE2 and anti-6-keto PGF1 alpha-antibodies, which prevent part of the thymulin effect, or 2) by simultaneous addition of PG and thymulin, at concentrations far lower than those which correspond to their thymulin-like effect. Moreover, PGE2 addition, at concentration close to that found to be endogenously produced, partially reversed the indomethacin-induced effect in the rosette assay. In conclusion, if PGs do not act as mediators of thymulin, they are able to synergize in one of its biological action.     

PG0026 is the C-terminal signal peptidase of a novel secretion system of Porphyromonas gingivalis

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) of ～70-80 amino acid residues that is essential for their secretion and attachment to the cell surface. The CTD itself has not been detected in mature substrates, suggesting that it may be removed by a novel signal peptidase. More than 10 proteins have been shown to be essential for the proper functioning of the secretion system, and one of these, PG0026, is a predicted cysteine proteinase that also contains a CTD, suggesting that it may be a secreted component of the secretion system and a candidate for being the CTD signal peptidase. A PG0026 deletion mutant was constructed along with a PG0026C690A targeted mutant encoding an altered catalytic Cys residue. Analysis of clarified culture fluid fractions by SDS-PAGE and mass spectrometry revealed that the CTD was released intact into the surrounding medium in the wild type strain, but not in the PG0026 mutant strains. Western blot experiments revealed that the maturation of a model substrate was stalled at the CTD-removal step specifically in the PG0026 mutants, and whole cell ELISA experiments demonstrated partial secretion of substrates to the cell surface. The CTD was also shown to be accessible at the cell surface in the PG0026 mutants, suggesting that the CTD was secreted but could not be cleaved. The data indicate that PG0026 is responsible for the cleavage of the CTD signal after substrates are secreted across the OM.     

Characterization of pollen polygalacturonase encoded by several cDNA clones in maize

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes form a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.     

Thymic hormones and prostaglandins II. Synergistic effect on mouse spontaneous rosette forming cells

Prostaglandins (PGs) have been assummed to play a role in the biological activity of thymic hormones (TH). Indeed, it has been shown that type E-PGs are able to mimic the action of several TH. Moreover, indomethacin interferes in the rosette assay, which still represents the most commonly used bioassay for the evaluation of TH and, in particular thymulin levels, in biological fluids. Previously, our attempt to modulate PG production by different TH showed that none of the TH tested affect PGE2, 6-keto-PGF1α, PGF2α and TXB2 production by spleen cells from control and thymectomized (Tx) mice, while indomethacin was able to inhibit the spontaneous PG production. Here, we investigated a possible role for each endogenously produced PG in the experimental conditions of the rosette assay, in order to define : 1) whether or not there was a specificity of action of a given PG ; and 2) to analyze the pattern of action between thymulin and the endogenously produced PGs. We demostrated that PE2 and 6-keto-PGF1α are the PGs which are physiologically involved in the rosette assay, according to their levels of endogeneous production, and that they are able to synergize with thymulin. This synergy was demonstrated in two ways: 1) by adding anti-PGE2 and anti-6-keto PGF1α-antibodies, which prevent part of the thymulin effect, or 2) by simultaneous addition of PG and thymulin, at concentrations far lower than those which correspond to their thymulin-like effect. Moreover, PGE2 addition, at concentration close to that found to be endogenously produced, partially reversed the indomethacin-induced effect in the rosette assay. In conclusion, if PGs do not act as mediators of thymulin, they are able to synergize in one of its biological action.     

Influence of stage of oestrous cycle on time of oestrus following cloprostenol treatment in the bovine

The first of 2 injections of 0.5 mg cloprostenol (PG1 and PG2) eleven days apart was given to 19 Friesian-Hereford cross heifers between days 8-14 of their cycle (Treatment A) and 16 similar animals between days 0-4 (Treatment B). Oestrus show was monitored by Kamar Heat Mount detectors and vasectomised bulls with chin-ball markers. Blood samples taken at PG1, six days later, at PG2 and four days later were assayed for progesterone to confirm that luteolysis had occurred as expected. Four hourly rectal examinations of the ovaries were carried out from 56-112 hours after PG2 and four hourly blood samples from 36-96 hours after PG2 were collected for FSH and LH assay. Mean time in hours from PG2 to oestrus onset, LH peak and ovulation respectively was 57.4 +/- 2.9, 60.2 +/- 2.0, 91.7 +/- 1.8 for Treatment A and 64.9 +/- 4.1, 68.9 +/- 2.4, 96.7 +/- 1.3 for Treatment B. Treatment A animals showed significantly higher (p<0.01) FSH levels at PG2 than Treatment B. Time from PG2 to LH peak was significantly shorter in animals treated either on days 7 and 8 (p<0.01) or days 15-16 (p<0.05) of their cycle compared with treatment on days 12-14 and it is suggested that these shorter response times correspond to an early and late cycle wave of follicular growth. Secondary FSH peaks some 28 hours after that occurring synchronously with the pre-ovulatory LH peak were observed to be significantly (p<0.01) higher at oestrus associated with the early cycle follicular growth wave as compared with that later in the cycle which may argue a difference in endocrine control of the two periods of follicular maturation.     

Polygalacturonase Isozymes and Pectin Depolymerization in Transgenic rin Tomato Fruit

We have previously described the construction and expression of a chimeric gene that allows developmentally regulated expression of tomato (Lycopersicon esculentum) polygalacturonase in ripening-impaired, mutant (rin) tomato fruit (JJ Giovannoni, D DellaPenna, AB Bennett, RL Fischer [1989] The Plant Cell 1: 53-63). We now show that expression of the chimeric polygalacturonase gene in rin tomato fruit resulted in the accumulation of all three polygalacturonase isozymes (PG1, PG2A, and PG2B). Polyuronide solubilization and polyuronide depolymerization both reached their maximal levels in transgenic rin fruit prior to the appearance of PG2 isozymes. These results demonstrate that PG1, PG2A, and PG2B all arise by differential processing of a single gene product and further suggest that the PG1 isozyme is sufficient to carry out both polyuronide solubilization and depolymerization in vivo.     

Channeling of eukaryotic diacylglycerol into the biosynthesis of plastidial phosphatidylglycerol

Plastidial glycolipids contain diacylglycerol (DAG) moieties, which are either synthesized in the plastids (prokaryotic lipids) or originate in the extraplastidial compartment (eukaryotic lipids) necessitating their transfer into plastids. In contrast, the only phospholipid in plastids, phosphatidylglycerol (PG), contains exclusively prokaryotic DAG backbones. PG contributes in several ways to the functions of chloroplasts, but it is not known to what extent its prokaryotic nature is required to fulfill these tasks. As a first step toward answering this question, we produced transgenic tobacco plants that contain eukaryotic PG in thylakoids. This was achieved by targeting a bacterial DAG kinase into chloroplasts in which the heterologous enzyme was also incorporated into the envelope fraction. From lipid analysis we conclude that the DAG kinase phosphorylated eukaryotic DAG forming phosphatidic acid, which was converted into PG. This resulted in PG with 2-3 times more eukaryotic than prokaryotic DAG backbones. In the newly formed PG the unique Delta3-trans-double bond, normally confined to 3-trans-hexadecenoic acid, was also found in sn-2-bound cis-unsaturated C18 fatty acids. In addition, a lipidomics technique allowed the characterization of phosphatidic acid, which is assumed to be derived from eukaryotic DAG precursors in the chloroplasts of the transgenic plants. The differences in lipid composition had only minor effects on measured functions of the photosynthetic apparatus, whereas the most obvious phenotype was a significant reduction in growth.     

Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments

DNA quantification of soils and sediments is useful for the investigation of microbial communities and for the acquisition of their genomes that are exploited for the production of natural products. However, in such samples DNA quantification is impaired by humic acids (HA). Due to its lack of specificity and sensitivity, UV spectrophotometry cannot be applied. Consequently, fluorimetric assays applying Hoechst (H) 33258 or PicoGreen (PG) are used. Here, we investigated the SYBR Green I (SG) assay, which was also affected by HA, but was found to be 25- and 1.7-fold more sensitive compared to the H 33258 and PG assays, respectively. Spectrophotometric, fluorimetric and quenching studies as well as gel mobility shift assays suggested that the effect of HA on the SG assay was based on an inner filter effect, collisional quenching and binding of SG to HA. As to the latter finding, the standard 6250-fold dilution of the SG reagent was optimised to a 2000-fold dilution. Although the sensitivity of the optimised SG assay was reduced by a factor of 1.3, the interfering effect of HA could be reduced up to 22-fold. A significant reduction of HA interferences by lowering the pH of the assay was not observed. Finally, the performance of the modified SG assay and the corresponding evaluation methods were verified by the determination of DNA recoveries and concentrations of standards and environmental samples in comparison to the PG assay.     

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.     

Activation of protein kinase C increases proteoglycan synthesis in immature rat Sertoli cells

In order to determine the signal transduction pathways involved in the regulation of proteoglycan (PG) synthesis in immature rat Sertoli cells (SC), we have examined the effect of the tumor promoter phorbol ester PMA (phorbol myristate acetate) on [35S]sulfate and [3H]glucosamine incorporation into PG molecules neosynthesized by cultured rat SC. PMA induced a dose- and time-dependent stimulation of labeled cell-associated PG as determined by quantitative solid phase assay. The overall effect of PMA resulted from enhancement of both glycosylation and catabolism of cell PG, this latter effect leading to a drastic decrease of their residence time in the membrane. Besides these quantitative effects, activation of protein kinase C by PMA induced qualitative changes as reflected by increase in relative proportion of heparan sulfate PG (HSPG) in cell membrane PG. In light of our previous results suggesting an inverse relationship between PG synthesis and FSH responsiveness in immature rat Sertoli cells, the PMA-induced upregulation of cell membrane PG, and particularly HSPG, could constitute one mechanism involved in the repression of FSH-stimulated steroidogenesis induced by PKC activation.     

Changes in prostaglandin concentration in blood subjected to repetitive freezing and thawing

Successive freezing and thawing of whole blood results in a consistently higher yield of various prostaglandin (PG) compounds. Evaluations were made with radioimmunological assay. The increase in PG concentrations seems to be more associated with cell fragmentation and not with the dissociation of albumin-PG complex. Our data suggest that there may be some dissociation of non-albumin-PG complexes. Artifactually high PG concentrations due to in vitro PG synthetase activity appears minimal at least with respect to indomethacin blocking of this enzyme. There are, in general, only slight differences in PG concentrations in samples with and without indomethacin.     

Changes in prostaglandin concentration in blood subjected to repetitive freezing and thawing.

Successive freezing and thawing of whole blood results in a consistently higher yield of various prostaglandin (PG) compounds. Evaluations were made with radioimmunological assay. The increase in PG concentrations seems to be more associated with cell fragmentation and not with the dissociation of albumin-PG complex. Our data suggest that there may be some dissociation of non-albumin-PG complexes. Artifactually high PG concentrations due to in vitro PG synthetase activity appears minimal at least with respect to indomethacin blocking of this enzyme. There are, in general, only slight differences in PG concentrations in samples with and without indomethacin.     

PG27 is a novel membrane protein essential for a Porphyromonas gingivalis protease secretion system

Porphyromonas gingivalis secretes endopeptidase gingipains, which are important virulence factors of this bacterium. Gingipains are transported across the inner membrane via the Sec system, followed by transport across the outer membrane via an unidentified pathway. The latter transport step is suggested to be mediated via a novel protein secretion pathway. In the present study, we report a novel candidate as an essential factor for the latter transport step. The PG0027 gene of P. gingivalis W83 encodes novel protein PG27. In a PG0027 deletion mutant (83K10), the activities of Arg-gingipain and Lys-gingipain were severely reduced, while the activities of secreted exopeptidases DPPIV, DPP-7, and PTP-A were unaffected. Protein localization was investigated by cell-surface biotinylation, subcellular fractionation, and immunoblot analysis. In the wild-type W83, Arg-gingipains in membrane fraction were detected as cell surface proteins. In contrast, in 83K10, Arg-gingipains were trapped in the periplasm and hardly secreted into an extracellular milieu. PG27 was suggested to be exposed to the cell surface by a cell surface biotinylation experiment; however, PG27 was detected in both inner and outer membrane fractions by subcellular fractionation experiments. Taken together, we suggest that PG27 is a unique membrane protein essential for a novel secretion pathway.