Peptidoglycan and lipopolysaccharide bind to the same binding site on lymphocytes

Bacterial cell wall peptidoglycan (PGN) and lipopolysaccharide (LPS), which are both macrophage activators and polyclonal B cell mitogens, were shown to bind to the same dominant 70-kDa 6.5 pI protein on the surface of mouse B lymphocytes. This conclusion was supported by the following results: (a) the PGN- and LPS-binding proteins co-migrated following photoaffinity cross-linking and two-dimensional polyacrylamide gel electrophoresis; (b) cross-linking of PGN to this 70-kDa protein was competitively inhibited by LPS (IC50 = 7.3 microM), LPS from a deep rough mutant (IC50 = 6.9 microM), and lipid A (IC50 = 18-72 microM); (c) cross-linking of LPS to this 70-kDa protein was competitively inhibited by polymeric soluble PGN (IC50 = 0.09 microM) and sonicated high Mr PGN (IC50 = 0.6 microM); (d) cross-linking of both PGN and LPS to this 70-kDa protein was also competitively inhibited by dextran sulfate (IC50 = 115-124 microM); (e) cross-linking of both PGN and LPS to this 70-kDa protein was inhibited by a (GlcNAc)2-specific lectin; and (f) peptide maps of the 70-kDa proteins digested with chymotrypsin, subtilisin, staphylococcal protease V, or papain were identical for PGN- and LPS-binding proteins and unique for each enzyme. Based on competitive inhibition experiments, binding of PGN to the 70-kDa protein was 20-1200 times stronger than the binding of LPS or lipid A on a per mol basis. However, when aggregated micellar structures of LPS or lipid A were considered, the avidities of LPS and PGN binding were similar. These results demonstrate binding of PGN and LPS to the same 70-kDa protein on lymphocytes and suggest that the binding is specific for the (GlcNAc-MurNAc)n backbone of PGN and the (GlcNAc)2 part of lipid A.     

Inhibitory effect of mitoxantrone on activity of protein kinase C and growth of HL60 cells.

Mitoxantrone, a new anthraquinone, showed inhibitory an effect on protein kinase C (PKC) activity. Its IC50 value was 4.4 micrograms/ml (8.5 microM), which is much lower than those of the well-known anthracyclines daunorubicin and doxorubicin, the IC50 values of which are more than 100 micrograms/ml (> 170 microM). Kinetic studies demonstrated that mitoxantrone inhibited PKC in a competitive manner with respect to histone H1, and its Ki value was 6.3 microM (Ki values of daunorubicin and doxorubicin were 0.89 and 0.15 mM, respectively), and in a non-competitive manner with respect to phosphatidylserine and ATP. Inhibition of phosphorylation by mitoxantrone was observed with various substrates including S6 peptide, myelin basic protein and its peptide substrate derived from the amino-terminal region. Their IC50 values were 0.49 microgram/ml (0.95 microM), 1.8 micrograms/ml (3.5 microM), and 0.82 microgram/ml (1.6 microM), respectively. Mitoxantrone did not markedly inhibit the activity of cyclic AMP-dependent protein kinase, casein kinase I or casein kinase II, at concentrations of less than 10 micrograms/ml. On the other hand, brief exposure (5 min) of HL60 cells to mitoxantrone caused the inhibition of cell growth with an IC50 value of 52 ng/ml (0.1 microM). In HL60 cells, most of the PKC activity (about 90%) was detected in the cytosolic fraction. When HL60 cells exposed to 10 micrograms/ml mitoxantrone for 5 min were observed with fluorescence microscopy, the fluorescence elicited from mitoxantrone was detected in the extranuclear area. These results indicated that mitoxantrone is a potent inhibitor of PKC, and this inhibition may be one of the mechanisms of antitumor activity of mitoxantrone.     

Characterization of a scintillation proximity assay to detect modulators of transforming growth factor alpha (TGF alpha) binding.

A scintillation proximity assay (SPA) for transforming growth factor alpha (TGF alpha) using SPA beads coated with A431 membranes has been studied. Binding of TGF alpha to the beads was characteristic of a receptor interaction. A class of high-affinity receptors for [125I]-TGF alpha (Kd = 0.10-0.26 nM) was detected by competition studies between [125I]TGF alpha and cold TGF alpha and by analysis of association and dissociation rate constants. An antibody to the epidermal growth factor receptor (clone 528) inhibited binding of [125I]TGF alpha (IC50 = 0.20 micrograms/ml), but an anti-TGF alpha antibody (clone 134A-2B3) (less than 25 micrograms/ml) did not block binding. Suramin inhibited [125I]-TGF alpha binding (IC50 = 0.20 mM). The ether lipids 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine, and rac-lyso-platelet activating factor inhibited TGF alpha binding (IC50 values of 49, 69, and 57 microM, respectively). SPA is a convenient method for identifying agents that may act by interfering with TGF alpha binding.     

A novel nuclear protein which binds to G gamma and A gamma globin promoters and modulates hemoglobin synthesis in K562 cells.

Nuclear extract of human erythroleukemic cell line K562 contains a 70 kDa protein which is gradually reduced when cells are induced to express globin genes by 25 microM hemin. When globin synthesis was inhibited by cycloheximide (100 micrograms/ml) or Actinomycin D (1 microgram/ml), the disappearance of this protein was prevented. The 70 kDa nuclear protein exhibited strong binding to G gamma and A gamma globin promoters but not to beta-globin promoter. This suggests that this 70 kDa nuclear protein may be involved in the regulation of fetal globin gene expression.     

Chloride channels in mast cells: block by DIDS and role in exocytosis

In rat peritoneal mast cells, we have investigated the influence of the chloride transport blocker 4,4''-diisothiocyanato-stilbene-2,2''- disulfonic acid (DIDS) and the extracellular chloride concentration on the chloride current induced by intracellular application of cyclic AMP (cAMP) and on hexosaminidase secretion from intact cells stimulated with compound 48/80. The inhibition of the Cl-current by extracellular DIDS is voltage and time dependent. Upon depolarization from -10 to +70 mV, the outward current diminishes with millisecond kinetics. The size of the steady state current and the time constant of the decrease both decrease with increasing DIDS concentrations. The steady state current at +70 mV is blocked by DIDS with an IC50 of 2.3 microM. The number of open channels at -10 mV is reduced with an IC50 of 22 microM. The electrophysiological and pharmacological properties of this current are most similar to those of the Cl- current in T lymphocytes activated by osmotic stress (Lewis, R. S., P. E. Ross, and M. D. Cahalan. 1993. Journal of General Physiology. 101:801-826). Extracellular DIDS also inhibits exocytosis. At optimal stimulation with 10 micrograms/ml compound 48/80 secretion is inhibited with an IC50 = 50 microM and a Hill coefficient n = 10. At half optimal stimulation with 1 microgram/ml inhibition occurs with an IC50 = 10 microM and n = 1. Substitution of extracellular chloride by glutamate has only very small effects on secretion stimulated with 10 micrograms/ml compound 48/80. We conclude that activation of the chloride current in mast cells is not essential for stimulation of exocytosis but may enhance secretion at suboptimal stimulation. Alternatively, the channel may play a role in volume regulation following degranulation.     

Bacterial peptidoglycan binds to tubulin

A search for cellular binding proteins for peptidoglycan (PGN), a CD14- and TLR2-dependent macrophage activator from Gram-positive bacteria, using PGN-affinity chromatography and N-terminal micro-sequencing, revealed that tubulin was a major PGN-binding protein in mouse macrophages. Tubulin also co-eluted with PGN from anti-PGN vancomycin affinity column and bound to PGN coupled to agarose. Tubulin-PGN binding was preferential under the conditions that promote tubulin polymerization, required macromolecular PGN, was competitively inhibited by soluble PGN and tubulin, did not require microtubule-associated proteins, and had an affinity of 100-150 nM. By contrast, binding of tubulin to lipopolysaccharide (LPS) had 2-3 times lower affinity, faster kinetics of binding, and showed positive cooperativity. PGN enhanced tubulin polymerization in the presence of 4 M glycerol, but in the absence of glycerol, both PGN and LPS decreased microtubule polymerization. These results indicate that tubulin is a major PGN-binding protein and that PGN modulates tubulin polymerization.     

Effect of BI-L-239, A-64077 and MK-886 on leukotriene B4 synthesis by chopped guinea pig lung and on antigen-induced tracheal contraction in vitro.

The 5-lipoxygenase (5-LO) inhibitors BI-L-239 and A-64077 were compared with the 5-LO translocation inhibitor MK-886 for the ability to inhibit leukotriene B4 (LTB4) biosynthesis by chopped (1 mm3) guinea pig lung. LTB4 synthesis by ovalbumin-sensitized chopped lung tissue was determined after stimulation with either calcium ionophore (A23187) or antigen. With A23187 stimulation, MK-886 was more potent (IC50 = 0.39 +/- 0.23 microM, mean +/- SEM, p < 0.01) than BI-L-239 (IC50 = 2.48 +/- 0.46 microM) or A-64077 (IC50 = 4.68 +/- 0.70 microM) and BI-L-239 was more potent than A64077 (p < 0.02). Thus, the order of potency was MK-886 > BI-L-239 > A-64077 for inhibition of calcium ionophore-induced LTB4 generation. There was no significant differences in potency of the compounds in chopped lung stimulated with antigen: IC50 for LTB4 synthesis by A-64077 = 3.31 +/- 1.70 microM, for BI-L-239 = 9.06 +/- 4.94 microM, and for MK-886 = 13.33 +/- 7.91 microM. The ability of these compounds to inhibit contraction of tracheal tissue from actively sensitized guinea pigs in response to antigen was also determined in the presence of indomethacin (15 micrograms/ml), mepyramine, and atropine (5 micrograms each/ml). Both 5-LO inhibitors inhibited antigen-induced contraction, with IC50 values for BI-L-239 and A-64077 of 1.58 and 4.35 microM respectively. MK-886 was ineffective at inhibiting antigen-induced tracheal contraction in vitro at concentrations up to 30 microM. In summary, these compounds inhibit antigen-induced and A23187-induced leukotriene biosynthesis in guinea pig tissue. These 5-LO inhibitors were similarly effective at inhibiting antigen-induced tracheal contraction where MK-886 was ineffective.     

The mechanism of action of DuP 721, a new antibacterial agent: effects on macromolecular synthesis

Pulse labeling studies with Bacillus subtilis showed that DuP 721 inhibited protein synthesis. The IC50 of DuP 721 for protein synthesis was 0.25 micrograms/ml but it was greater than 32 micrograms/ml for RNA and DNA synthesis. In cell-free systems, DuP 721 concentrations up to 100 microM did not inhibit peptide chain elongation reactions under conditions where chloramphenicol, tetracycline and hygromycin B inhibited these reactions. Furthermore, Dup 721 did not cause phenotypic suppression of nonsense mutations suggesting that DuP 721 did not inhibit peptide chain termination. Thus, the mechanism of action of DuP 721 is at a target preceeding chain elongation.     

Cholinergic-induced [3H] noradrenaline release in rat brain cortical slices is mediated via a pertussis toxin sensitive GTP binding protein and involves activation of protein kinase C

The involvement of a GTP-binding protein (G-protein) in the process of neurotransmitter release was examined using pertussis toxin and cholera toxin. Cholinergic agonists are shown to mediate [3H]noradrenaline release in rat brain slices via a pertussis toxin (1.2 micrograms/ml) sensitive, and cholera toxin (0.5 microgram/ml) insensitive G-protein. An indication for the involvement of a G-protein and phospholipase C activation in the release process was implied from the inhibitory effect of neomycin on K+-, veratridine- and carbachol-induced-norepinephrine release. Depolarizing agents mediate a neomycin-sensitive release, which is not which is not affected either by pertussis toxin or cholera toxin, suggesting a different mode of phospholipase C activation, unlike carbachol-induced release, which is both neomycin and pertussis toxin sensitive. Similarly, a hormone-sensitive carrier activated by phenylephrine not via alpha 1-adrenergic receptors, mediates a non-exocytosis efflux which is not affected by neomycin and is shown to be pertussis toxin-insensitive. The inhibitory action of protein kinase C inhibitors polymyxin B, K252a and H-7 [(1-(5-isoquinolinesulphonyl)-2-methyl-piperazine] on release, strongly suggests its participation in the process. Polymyxin B, a relatively selective protein kinase C inhibitor, inhibited carbachol-induced release (IC50 = 0.53 microM) as well as the K+ and the veratridine induced [3H] noradrenaline release, K252a, an inhibitor of various protein kinases at the ATP site, and H-7, another protein kinase C inhibitor, inhibited carbachol-induced noradrenaline released with IC50 = 35 nM and 3 microM respectively. Consistent with its inability to activate phospholipase C, phenylephrine-induced noradrenaline efflux was unaffected by polymyxin B (greater than 70 microM). These results offer more supportive evidence for a major role played by the dual messengers inositol trisphosphate and diacylglycerol (IP3/DG) in the mechanisms of neuronal release.     

Norlignans from Sequoia sempervirens

Six new norlignans, named sequosempervirins B-G (1-6), together with three known norlignans, agatharesinol (7), agatharesinol acetonide (8), and sugiresinol (9), were isolated from the branches and leaves of Sequoia sempervirens. Their structures were determined mainly by high-resolution mass spectroscopy (HR-MS), and various 1D- and 2D-NMR methods, as well as, in the case of 1, by means of X-ray diffraction. Compound 8 showed anticancer activity towards the A549 non-small-cell lung-cancer cell line (IC50 = 27.1 microM). The acetone extract of S. sempervirens was found to be antifungal towards Candida glabrata (IC50 = 15.98 microg/ml), and both the acetone and MeOH extracts inhibited the proteolytic activity of cathepsin B (IC50 = 4.58 and 5.49 microg/ml, resp.).     

Glycoprotein biosynthesis in B lymphocytes: induction of protein N-glycosylation, RNA synthesis, and DNA synthesis by phorbol ester plus ionomycin is blocked by protein kinase inhibitors

The combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin produces a dramatic increase in the incorporation of [2-3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and glycoprotein, and the induction of RNA and DNA synthesis in murine splenic B lymphocytes (B cells). The kinetics of the induction processes and the concentrations of PMA and ionomycin required for the optimal response have been defined. While the levels of induction of RNA and DNA synthesis by PMA + ionomycin were similar to the mitogenic response to bacterial lipopolysaccharide, activation by PMA and the calcium ionophore resulted in a threefold higher stimulation in dolichol-linked oligosaccharide biosynthesis and protein N-glycosylation. These results indicate that all signalling mechanisms that trigger RNA and DNA synthesis may not be sufficient to produce maximal induction of the N-glycosylation apparatus. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor, prevented the induction of protein N-glycosylation activity (IC50 = 11 microM), as well as RNA (IC50 = 18 microM) and DNA synthesis (IC50 = 12 microM), two common indices of B cell activation. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) also inhibited the induction of oligosaccharide-lipid intermediate, glycoprotein, RNA, and DNA synthesis, but required higher concentrations than H-7 for 50% inhibition. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a potent inhibitor of cyclic nucleotide-dependent protein kinases, had little effect on the activation of the B cell metabolic processes. The H-7-sensitive reactions involved in the induction of RNA and DNA synthesis occurred within 4 h, but induction of lipid intermediate and glycoprotein biosynthesis remained sensitive to H-7 for 10 h after exposure to PMA and ionomycin. Direct in vitro assays in the presence of 0.6% Brij 58 reveal that a cytosolic, phospholipid-dependent protein kinase activity is translocated to a membrane site(s) after treatment with PMA and ionomycin, and the translocated protein kinase is sensitive to H-7. The relative order of potency of the protein kinase inhibitors on the metabolic processes strongly supports the hypothesis that protein kinase C, acting synergistically with Ca2+ mobilization, plays a key regulatory role in the early stages of B cell activation. The synthesis of oligosaccharide-lipid intermediates and protein N-glycosylation are also shown to be induced in B cells activated by PMA + ionomycin.     

Partial purification and characterization of DNA-dependent RNA polymerases I and II from cherry salmon (Oncorhynchus masou)

1. DNA-dependent RNA polymerases I and II were purified approx 3900- and 13,000-fold, respectively, from sonicated nuclear extract of cherry salmon (Oncorhynchus masou) liver by DEAE-Sephadex, heparin-Sepharose and DNA-cellulose column chromatography. 2. The purified RNA polymerases exhibited a requirement for four kinds of ribonucleoside 5'-triphosphates, an exogeneous template and divalent cation. 3. The activities of RNA polymerases I and II were inhibited by Actinomycin D (24 micrograms/ml) but not by Rifampicin (200 micrograms/ml). 4. RNA polymerase I preferred native DNA as template, while polymerase II preferred single-stranded DNA. 5. RNA polymerase II was inhibited by a low concentration of alpha-amanitin (0.02 micrograms/ml). RNA polymerase I was also inhibited by the relatively high concentration of alpha-amanitin (IC50 = 100 micrograms/ml and IC70 = 750 micrograms/ml). 6. RNA polymerases from cherry salmon exhibited a higher activity at low temperature than from rat liver.     

The inhibition of phosphatidylinositol 3-kinase by quercetin and analogs.

Phosphatidylinositol (PtdIns) 3-kinase is an enzyme involved in cellular responses to growth factors. Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyrano-4-one), a naturally occuring bioflavinoid, was found to inhibit PtdIns 3-kinase with an IC50 of 1.3 micrograms/ml (3.8 microM); inhibition appears to be directed towards the ATP binding site of the kinase. Analogs of quercetin were also investigated as PtdIns 3-kinase inhibitors, with the most potent compounds exhibiting IC50's in the range of 1.7-8.4 micrograms/ml (5-19 microM). In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 greater than 30 micrograms/ml). These findings suggest that flavinoids may serve as potent inhibitors of PtdIns 3-kinase. Furthermore, the enzyme is much more sensitive to substituents at the 3-position of the flavinoid ring than are other protein and PtdIns kinases, suggesting that specific inhibitors of PtdIns 3-kinase can be developed to explore the biological role of the enzyme in cellular proliferation and growth factor response.     

The role of a GTP-binding protein in coupling of a muscarinic cholinergic receptor and Na,K-ATPase in myocardial sarcolemma]

The effects of the cholinergic agonist carbachol (Cch) and guanine nucleotides on the Na,K-ATPase and K-dependent p-nitrophenylphosphatase (K-p-NPPase) activities in rabbit and dog myocardial sarcolemma vesicles in the presence of the pore-forming antibiotic alamethicin (20 micrograms/ml), was studied. Cch (0.01-100 microM) inhibited the both enzymatic activities by 40-45% (IC50 = 0.3-0.5 microM) only after addition of GTP (50 microM) or its analogs: GTP gamma S (0.1-1.0 microM) and Gpp(NH)p (10 microM). The muscarinic acetylcholine receptor (mAchR) antagonist atropine (10 microM) blocked the effect of Cch. GTP gamma S alone produced a concentration-dependent decrease in the both Na,K-ATPase and K-p-NPPase activities by 40-45% (IC50 = 1-2 microM) with a lag period of about 3 minutes; this lag disappeared in the presence of the agonist. The GDP analog GDP beta S (0.01-100 microM) neither affected these activities nor promoted the inhibiting effect of Cch. Pretreatment of sarcolemmal vesicles with 20 micrograms/ml of pertussis toxin in the presence of 100 microM NAD abolished the inhibiting effect of Cch on the Na,K-ATPase and phosphatase activities. Under these conditions pertussis toxin catalyzed the ADP-ribosylation of alpha-subunits of the inhibitory GTP-binding protein (G1) which were identified immunochemically as alpha i2, alpha i3 and, possibly, alpha i1. The data obtained testify to the involvement of G1 in the mAchR-mediated inhibition of myocardial sarcolemmal Na,K-ATPase as well as in the signal transduction from the receptor to the enzyme.     

Structural studies on molecular interactions between camel peptidoglycan recognition protein, CPGRP-S, and peptidoglycan moieties N-acetylglucosamine and N-acetylmuramic acid

Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.     

D-mannonolactam amidrazone. A new mannosidase inhibitor that also inhibits the endoplasmic reticulum or cytoplasmic alpha-mannosidase.

The amidrazone of D-mannonolactam (see compound 5, Fig. 1) was synthesized chemically as a mimic of the mannopyranosyl cation and tested as a potential inhibitor of mannosidases. In this study compound 5 is shown to be a more general mannosidase inhibitor than other currently known compounds and exhibits properties not previously observed with any other mannosidase inhibitors. Thus D-mannonolactam amidrazone not only inhibits the Golgi mannosidase I (IC50 = 4 microM) and mannosidase II (IC50 = 90-100 nM), but it is the first inhibitor that has been shown to be a potent inhibitor of the soluble or endoplasmic reticulum alpha-mannosidase (IC50 = 1 microM). This compound also inhibited the aryl-mannosidases regardless of anomeric configuration although it was much more effective on enzymes recognizing alpha-linked mannose, i.e. jack bean and mung bean alpha-mannosidases (IC50 = 400 nM) as compared with fungal beta-mannosidase (IC50 = 150 microM). Mannonoamidrazone was tested in animal cell cultures using influenza virus-infected Madin-Darby canine kidney cells as a model system, and was found to prevent almost completely the formation of complex types of N-linked oligosaccharides with the formation of about equal amounts of Man9(GlcNAc)2 and Man8(GlcNAc)2 structures. Thus D-mannonolactam amidrazone is a potent but broad spectrum mannosidase inhibitor whose structure and properties should provide valuable insight into the design of other useful glycosidase inhibitors.     

Equilibrium Binding Analysis of Neural Cell Adhesion Molecule Binding to Heparin

The kinetics of neural cell adhesion molecule (NCAM) binding to heparin were studied in a heparin-Sepharose-based solid-phase binding assay. The observed binding is time dependent and saturable. A binding constant of 5.2 +/- 1.4 X 10(-8) M is observed for binding of newborn rat NCAM to heparin. This is approximately 25 times lower than the binding constant determined for newborn rat NCAM homophilic binding. Both Scatchard and Hill plot analyses suggest the presence of only one binding site. Fab' fragments of antibodies to rat NCAM significantly inhibit binding, a result indicating that a specific site on NCAM is involved in binding to heparin. The binding is inhibited by heparin (IC50, approximately 5 micrograms/ml), whereas chondroitin sulfate is a less potent inhibitor (IC50, approximately 15 micrograms/ml).     

Effect of heparin on proliferation and signalling in BC3H-1 muscle cells. Evidence for specific binding sites

We studied binding and growth inhibitory properties of different glycosaminoglycans in growing and differentiated BC3H-1 muscle cells. Heparin (10 micrograms/ml) and heparan sulfate (10 micrograms/ml) significantly inhibited DNA synthesis in growing and differentiated cells, as monitored by [3H]thymidine incorporation. Binding of heparin to BC3H-1 cells was specific and time-dependent. Heparan sulfate was the only glycosaminoglycan able to displace [3H]heparin (IC50, 3.2 x 10(-7) M), although it was 10-fold less effective than heparin itself (IC50, 3.6 x 10(-8) M). Scatchard analysis revealed the existence of high-affinity heparin binding sites (Kd, 5 x 10(-8) M). Furthermore, heparin inhibited serum-induced stimulation of inositol lipid turnover. Taken together, these results indicate that heparin inhibits BC3H-1 cell growth by interacting with the cell surface, possibly disrupting the flow of growth factor-related mitogenic signalling.     

The inhibitory effects of okadaic acid on platelet function.

Okadaic acid (OA), a potent inhibitor of protein phosphatases type 1 and type 2A, inhibited thrombin-induced platelet aggregation (IC50 = 0.8 microM), [14C]serotonin release and increase in intracellular Ca2+ ([Ca2+]i) in the same dose dependence. In the absence of thrombin OA increased the phosphorylation of 50-kDa protein and 20-kDa myosin light chain (MLC20). The 50-kDa protein phosphorylation was accomplished within a shorter time period and at a lower concentration than was the MLC20. OA decreased the thrombin-induced phosphorylation of 47-kDa protein and MLC20, although phosphorylation of MLC20 reincreased at higher concentrations of OA (5-10 microM). Since type 2A phosphatase is more sensitive to OA than type 1, these results suggest that type 2A phosphatases are involved in the regulation of Ca2+ signaling in thrombin-induced platelet activation.     

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.