Sprouty2 inhibition promotes proliferation and migration of periodontal ligament cells.

OBJECTIVES: We previously demonstrated that a dominant-negative Sprouty2 (Spry2) mutation promotes osteoblast proliferation and differentiation after basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulation, whereas it diminishes proliferation of gingival epithelial cells, thereby inducing favourable conditions for periodontal tissue regeneration. In this study, we investigated how Spry2 inhibition affects the cellular physiology of periodontal ligament (PDL) cells. METHODS: A total of 1-17 PDL cells (multipotent clonal human PDL cell line) were stimulated with bFGF and EGF after transfection of Spry2 siRNA. Cell proliferation, migration, ALP staining, real-time PCR, Western blot and immunofluorescence assays were performed. RESULTS: ERK1/2 activation and proliferation of 1-17 PDL cells were significantly upregulated by the addition of Spry2 siRNA in the presence of bFGF and EGF. In addition, Spry2 siRNA reduced transcription of osteogenesis-related genes and ALP staining relative to control cells. Furthermore, it increased AKT/phosphatidylinositol 3-kinase (PI3K) phosphorylation; consequently, Rac1 but not Cdc42 was activated, thereby promoting lamellipodia formation, cell proliferation and migration after stimulation by bFGF and EGF. CONCLUSION: Spry2 combined with bFGF and EGF stimulation reduced PDL cell migration and proliferation with inducing osteoblastic differentiation. These in vitro findings may provide a molecular basis for novel therapeutic approaches for establishing periodontal tissue regeneration.

The effects of vasoactive agents, platelet agonists and anticoagulation on thrombelastography.

BACKGROUND: Platelet activation is a critical step in primary hemostasis and clot formation. We tested a hypothesis that platelet stimulating effects of vasoactive agents or platelet agonists could be shown using thrombelastography (TEG) as faster onset or increased clot strength. We further examined if TEG could be modified to evaluate activated platelets as a reversal of anticoagulation in the presence of partial thrombin inhibition. METHODS: Blood samples were obtained from 126 non-cardiac surgical patients. Effects of vasoactive agents on TEG and aggregometry were examined using epinephrine, norepinephrine, vasopressin, desmopressin acetate, milrinone and olprinone (Experiment I). Platelet agonists (epinephrine, ADP and collagen) were separately tested on TEG (Experiment II). Effects of platelet agonists (ADP and collagen) on TEG under anticoagulation in the absence or presence of abciximab were studied (Experiment III). We also tested antiplatelet effects of milrinone and olprinone in the presence of anticoagulants on TEG (Experiment IV). RESULTS: Neither vasoactive agents nor platelet agonists affected TEG or aggregometry results except for milrinone and olprinone on aggregometry (Experiment I, II). Platelet agonists facilitated clotting in the presence of anticoagulants (Experiment III). Abciximab-treated platelets still exhibited procoagulant effects in the presence of heparin, while not in the presence of argatroban (Experiment III). Platelet inhibition on the modified TEG was more extensive with milrinone than olprinone, and it was dose dependent (Experiment IV). CONCLUSION: Modified TEG using heparin or argatroban might delineate the procoagulant effects of platelets by adding platelet specific agonist.

Identification of a dominant negative mutant of Sprouty that potentiates fibroblast growth factor- but not epidermal growth factor-induced ERK activation.

Various mitogenic stimuli such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and phorbol 12,13-dibutyrate (PDBu) activate the Ras-Raf-MEK-ERK pathway, but the regulatory mechanism of this pathway remains to be investigated. Here we found that in 293 cells, mammalian Sprouty2 and Sprouty4 were rapidly induced by EGF, FGF, and PDBu in an ERK pathway-dependent manner. Forced expression of Sprouty2 and Sprouty4 inhibited FGF-induced ERK activation but did not affect EGF- or PDBu-induced ERK activation. To examine whether endogenous Sproutys were also selective inhibitors, we generated a dominant negative form of Sprouty2 (Y55A) and Sprouty4 (Y53A) in which conserved tyrosine residues were mutated. These mutants reverted the suppressive effect of both Sprouty2 and Sprouty4 but not that of RasGAP or SPRED (Sprouty-related EVH1 domain-containing protein), another Sprouty-related Ras suppressor. Expression of dominant negative Sprouty2 and Sprouty4 enhanced and prolonged FGF- but not EGF-induced ERK activation in 293 cells. In PC12 cells, endogenous Sprouty4 was also induced by FGF. Overexpression of wild-type Sprouty4 blocked FGF-induced differentiation, whereas Y53A-Sprouty4 enhanced it. These observations suggest that endogenous Sprouty2 and Sprouty4 are physiological negative feedback regulators of growth factor-mediated ERK pathway and that there are Sprouty-sensitive and -insensitive ERK activation pathways. Finding a dominant negative form of Sproutys will facilitate the study of the molecular mechanism and physiological function of Sproutys.

Confirmation of minor components of less polar neutral and acidic glycolipids in monkey brain tissue.

Crude glycolipids, prepared without alkali treatment in advance, were separated into neutral and acidic glycolipids by DEAE-Sephadex A-25 (acetate form) column chromatography. Each glycolipid was further fractionated by a Silica gel 60-column chromatography. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS) of the intact glycolipid fractions, the less polar neutral glycolipids were found to contain alkali-labile ester cerebrosides and Galb-1-Diradylglycerols, whereas the less polar acidic glycolipids were found to contain alkali-labile ester sulfatide, HSO(3)-3Gal-1-Diradylglycerols, and novel alkali-stable plasmalo-sulfatides and ester or plasmalo HSO(3)-3Galb-1-Diradylglycerols as minor components of glycolipids in monkey brain tissue. In conclusion, minor components of less polar neutral and acidic glycolipids in monkey brain tissue were confirmed as ester cerebrosides, Galb-1-Diradylglycerols, ester sulfatides, HSO(3)-3Galb-1-Diradylglycerols, and novel plasmalo-sulfatides and ester or plasmalo HSO(3)-3Galb-1-Diradylglycerols by DE MALDI-TOF MS.

Isolation of reducing oligosaccharide chains from the chondroitin/dermatan sulfate-protein linkage region and preparation of analytical probes by fluorescent labeling with 2-aminobenzamide.

The glycosaminoglycan (GAG)-protein linkage regions of various proteoglycans share the common tetrasaccharide GlcA-Gal-Gal-Xyl-attached to Ser residues in the core proteins. In previous analysis we demonstrated unique modifications by epimerization, sulfation and phosphorylation of the component sugars. Here we developed a sensitive analytical method for the linkage region oligosaccharides to detect or monitor structural variations and changes. This will be useful for investigation of their biological roles, which are largely unknown, but they have been implicated in biosynthesis. A variety of linkage region-derived hexasaccharides was first prepared as reducing sugar chains from peptide chondroitin/dermatan sulfate of whale cartilage, shark cartilage, and bovine aorta by means of chondroitinase digestion in conjunction with beta-elimination in the absence of reducing reagents, but involving a mild alkali, 0.5 M LiOH, at 4 degrees C to prevent peeling reactions. The structures of these oligosaccharides were determined by the combination of HPLC, enzymatic digestion, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and (1)H NMR spectroscopy, which revealed eleven different hexasaccharides including a novel structure, DeltaHexAalpha1-3GalNAcbeta1-4IdoAalpha1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xyl (DeltaHexA and IdoA represent unsaturated hexuronic acid and L-iduronic acid, respectively). These oligosaccharides were labeled with a fluorophore, 2-aminobenzamide, to prepare analytical probes using the recently developed procedure [Kinoshita and Sugahara (1999) Anal. Biochem. 269, 367-378]. The fluorophore-tagged hexasacharides of low picomoles were well separated by HPLC and successfully analyzed by MALDI-TOF mass spectrometry. The principle of the method should be applicable to the analysis of the linkage region oligosaccharides derived from heparin and heparan sulfate as well.

Usefulness of MALDI/TOF mass spectrometry of immunoprecipitated serum variant transthyretin in the diagnosis of familial amyloid polyneuropathy.

A matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry (MS) system was used to detect variant transthyretin (TTR) in immunoprecipitated serum TTR molecules obtained from 6 patients with familial amyloid polyneuropathy (FAP) who were already proven not to have ATTR Val30Met. This simple and quick method showed six different patterns of mass spectra of TTR-related immunoprecipitates from these patients, and in each patient the clearly identified characteristic doublet-shaped ion peaks consisted of normal and variant TTR apart from each other peak with a mass difference between them. DNA sequencing confirmed that the patterns of variant TTR corresponded respectively to ATTR Val30Leu, ATTR Phe33Val, ATTR Asp38Ala, ATTR Ser50Arg, ATTR Ala97Gly and ATTR Ala97Ser. ATTR Asp38Ala and ATTR Ala97Ser are previously unknown variants of TTR leading to the development of FAP. ATTR Phe33Val was found in a Chinese FAP patient and ATTR Ala97Ser in a Taiwanese. Serum analysis using immunoprecipitation and MALDI/TOF MS system can provide useful information when investigating FAP patients with diverse types of variant TTR.

Application of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in tissues from sphingolipidosis patients.

Sphingolipidosis is due to defects in enzymes involved in hydrolysis of sphingolipids. We analyzed sphingolipids in tissues from patients with sphingolipidosis, including Farber disease (FD, acid ceramidase deficiency), Gaucher disease (GD), Niemann-Pick disease type C (NPDC), and GM1-gangliosidosis (GM1G), using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS). Crude lipids were extracted from about 100 mg wet weight of autopsied tissues, including liver, spleen, cerebrum or cerebellum. After mild alkaline treatment, a sphingolipid fraction was prepared from the crude lipids and analyzed by DE MALDI-TOF-MS. The results were as follows: (a) In FD liver both the ceramide/sphingomyelin and ceramide/monohexosylceramide ratios were significantly high; (b) in both liver and spleen from a GD patient, the glucosylceramide/sphingomyelin ratio was raised; (c) in liver from a NPDC patient, the monohexosylceramide/sphingomyelin ratio was markedly low, suggesting an increase of sphingomyelin; and (d) in all tissues examined in the GM1G patient, GM1-gangliosides or asialo-GM1-gangliosides, that are undetectable in a normal control, were increased. In conclusion, sphingolipids in human tissues could be directly determined by DE MALDI-TOF-MS, with only a small amount of specimens. This method will be useful for the diagnosis and biochemical evaluation of sphingolipidosis patients.

Contradictory functions of sulfatide in the blood coagulation system as coagulant and anticoagulant.

Sulfatide (galactosylceramide I3 -sulfate) has been reported to activate blood coagulation factor XII (Hageman factor), which suggests that it exhibits coagulant activity (Fujikama et al., 1980 Biochemistry 19, 1322-1330) However, sulfatide administered into animals as a bolus shot without subsequent thrombus formation, prolonged conventional clotting times and bleeding time (Hara et al., 1996 Glycoconjugate J. 13, 187-194). These findings suggest that it may exhibit anticoagulant rather than coagulant activity. Following this suggestion we found in vitro that binding of sulfatide to fibrinogen resulted in disturbance of fibrin formation. To examine a possible pharmacological effect of sulfatide on blood coagulation in vivo we continuously infused sulfatide into rats through plastic cannulae and found formation of giant thrombi around the tips of the cannulae. These data suggest that sulfatide may exhibit contradictory functions in the blood coagulation system.

Adult Sandhoff's disease: R505Q and I207V substitutions in the HEXB gene of the first Japanese case.

We describe a 31-year-old Japanese man with adult Sandhoff s disease presenting as spinocerebellar degeneration. There was a marked cerebellar atrophy on MRI, and proliferation of abundant PAS-positive foamy macrophages in the rectal mucosa. The activities of total beta-Hex, beta-Hex A, and beta-Hex B in leucocytes of the patient were 14%, 15%, and 6% of control values, respectively. However, oligosacchariduria or ultrastructural storage materials in liver tissue were nil. Direct sequencing of cDNA and genomic DNA, and restriction digestion revealed two different homozygous base substitutions in the HEXB gene: the G1514-->A substitution (R505Q) and the A619-->G substitution (1207V). The parents were consanguineous. His healthy mother, an enzymatic heterozygous carrier, was homozygous for 1207V, but heterozygous for R505Q mutation. Thus, the patient is probably homozygous for both base substitutions and a R505Q mutation may be linked to the phenotype of adult Sandhoff's disease.

Sulfatide can markedly enhance thrombogenesis in rat deep vein thrombosis model.

Although sulfatide (galactosylceramide I3-sulfate) has been reported to activate blood coagulation factor XII (Hageman factor), it has been administered to animals without subsequent thrombus formation. We recently found that sulfatide binds to fibrinogen and thus disturbs fibrin formation in vitro, suggesting its possible role as an anticoagulant rather than as a coagulant. We therefore examined the in vivo effects of sulfatide on thrombogenesis by using a rat deep vein thrombosis model in which thrombus is induced by ligating the inferior vena cava. Sulfatide and gangliosides were each separately administered to rats 1 min before the vein ligation, and after 3 h, sulfatide but not gangliosides markedly (P < .001) enhanced the thrombogenesis. A kinetic turbidmetric assay of plasma coagulation initiated by CaCl2 in the wells of a microtiter plate revealed that coagulation was also markedly accelerated in the presence of sulfatide but not gangliosides, the results of which seemed to be very consistent with those of the in vivo experiments. Because sulfatide could not induce thrombosis without vein ligation in rats, the enhancement of thrombogenesis by sulfatide in the in vivo model might require endothelial damage and/or venous congestion, both of which could be induced by vein ligation.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of glycosphingolipids including gangliosides.

Long chain base compositions of gangliosides containing mainly stearic acid could be determined without any chemical modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS). The analytical results for the long chain base compositions of various samples of GM1 from the brain tissues of patients with different diseases at different ages confirmed that the proportion of d20:1 (icosasphingosine) and d20 (icosa-sphinganine) of the total sphingosine bases increased quickly until adolescent or adult age and then remained constant slightly exceeding 50%; this value was evidently higher than the proportion of d20:1 and d20 of GM1 in various adult mammalian brains. A long chain base composition of GM1 from the brain tissue of a patient with infantile type of GM1-gangliosidosis at 4y2m was abnormal and so was in two sibling patients with Spielmeyer-Vogt type of juvenile amaurotic idiocy at 19y and 21y in spite of that in the latter there was no accumulation of GM1 in the brain tissue. On the other hand, a patient with adult type of GM1 gangliosidosis at 66y showed a local accumulation of GM1 in the putamen and caudate nucleus, but its long chain base composition was found to be normal. It was of interest that the white matter of Eker rat with hereditary renal carcinoma contained a large amount of plasmalocerebroside as compared with the amount of cerebroside and sphingomyelin. The individual molecular species of plasmalocerebroside were identified by DE MALDI-TOF MS.

Application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry with delayed ion extraction to ganglioside analyses.

Various monosialo- and disialo-gangliosides and their derivatives were examined by delayed ion extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF MS) in the reflector mode with alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid used as the matrix. Native gangliosides were generally found to give good spectra in the negative ion mode. 2,5-Dihydroxybenzoic acid was a better matrix for gangliosides than alpha-cyano-4-hydroxycinnamic acid, because this matrix seemed to minimize loss of sialic acid and carbon dioxide of gangliosides. About 1 pmol of ganglioside was able to be detected with this matrix. When "A-series" gangliosides such as GD1a and GalNAc-GD1a gave undesirable extra peaks probably due to loss of sialic acid besides molecule-related ion peaks, the methyl-esterification of the gangliosides at the carboxyl groups of sialic acids was found to be necessary to obtain good DE MALDI-TOF mass spectra in the positive ion mode. In contrast, "B-series" gangliosides such as GD1b, GD2, and GD3 gave rise to major dehydrated molecule-related ion [M-H2O-H]- peaks in the negative ion mode without the pretreatment of methyl-esterification. The DE MALDI-TOF mass spectrometric analysis enabled us to distinguish between GD1a and GD1b, which have the same molecular weight. It was also found that not only a purified sample, but also a mixed sample of various gangliosides was amenable to the identification of them by DE MALDI-TOF MS.

Preparation of various lysogangliosides including lyso-fucosyl GM1 and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis.

Our rapid method of microwave-mediated saponification for preparing lysoglycosphingolipids from their parent glycosphingolipids was also able to prepare lysogangliosides or modified lysogangliosides, which were identified by delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometric (DE MALDI-TOF MS) analysis. When GM3, GM2, and GM1 isolated from adult human brain gangliosides were subjected to the saponification, GM3 was found to give rise to only lyso-GM3 containing de-N-acetylneuraminic acid (de-N-acetyl lyso-GM3), whereas the GM2 produced both lyso-GM2 and the de-N-acetyl compound, and GM1 also gave both lyso-GM1 and the de-N-acetyl compound. In the saponification of GM1 and GDla, isolated from rat brain gangliosides, GM1 similarly produced both lyso-GM1 and the de-N-acetyl compound, but GDla was found to give rise to both dehydrated de-N-monoacetyl and dehydrated de-N-diacetyl lyso-GDla. However, the saponification of the GM1 fraction isolated from porcine brain gangliosides gave rise not only to both lyso-GM1 and the de-N-acetyl compound, but also unexpectedly to both lyso-fucosyl GM1 and its de-N-acetyl compound. The untreated GM1 fraction was examined by TLC and DE MALDI-TOF mass spectrometry, and proved to contain fucosyl-GM1. The DE MALDI-TOF MS analysis of the prepared lyso-gangliosides showed that their long chain bases consisted of d18:1 and d20:1 sphingosines in various ratios reflecting those of the different mammalian brain gangliosides.

Rapid method of preparation of lysoglycosphingolipids and their confirmation by delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

We have recently developed a rapid method for the preparation of lysoglycosphingolipids on a small scale, in high yield. This procedure of microwave-mediated saponification of about 1 mg of glycosphingolipid with 0.5 ml of 0.1 M NaOH in methanol for two minutes can be easily repeated if larger amounts of lyso-compounds are needed. We have also found that the new methodology of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the reflector mode is extremely effective for the confirmation of different lysoglycosphingolipids together with their long chain base components. The combined method of preparation and confirmation of lysoglycosphingolipids is also important for the identification of long chain bases of various sphingolipids, because the usual analytical method of long-chain bases of sphingolipids depends on acidic methanolysis, which results in the formation of by-products such as O-methylsphingosines and threo-sphingosines.

A microwave-mediated saponification of galactosylceramide and galactosylceramide I3-sulfate and identification of their lyso-compounds by delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Small amounts of galactosylceramide (cerebroside) and galactosylceramide I3-sulfate (sulfatide) obtained from porcine spinal cord and equine kidney were deacylated by a rapid method of microwave-mediated saponification to prepare their lyso-compounds. Mass spectra of their protonated or deprotonated molecular ion peaks were detected by recently developed new technology of a delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometer with reflector detector in positive or negative ion mode. Long chain bases of lysocerebroside and lysosulfatide were different between porcine spinal cord and equine kidney, but similar to each other in the same organ, suggesting their common synthetic pathway. It is noted that the new rapid method can be similarly applied to the deacylation of both cerebroside and sulfatide in contrast to our classical method which was able to be applied to cerebroside, but not to sulfatide.

Sulfatide prolongs blood-coagulation time and bleeding time by forming a complex with fibrinogen.

Sulfatides (galactosylceramide I3-sulfate), which are found in serum lipoproteins of various mammals, effectively increased prothrombin time (anticoagulant effect) and also effectively prolonged bleeding time (anti-platelet effect). When equal volumes of a homogeneous micellar solution of sulfatide and fibrinogen in phosphate-buffered saline were mixed, an insoluble complex precipitated. Analysis of the precipitated complex showed that the molar ratio of sulfatide to fibrinogen was about 400:1. These results indicate that the sulfatide micelle binds tightly to fibrinogen and thereby interferes with both fibrin gel formation (anticoagulant activity) and platelet function.

Suppression of Theiler's murine encephalomyelitis virus induced demyelinating disease by administration of gangliosides.

Intracerebral (i.c.) inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelinating disease. Gangliosides are membrane components of essentially all eukaryotic cells and are abundant in plasma membranes. Endogenous gangliosides have been implicated in cell recognition, cell adhesion, cell differentiation and neurite outgrowth. We studied the effect of gangliosides on TMEV-induced demyelinating disease (TMEV- IDD). We injected TMEV intracerebrally into susceptible SJL/J mice and induced TMEV-IDD. Gangliosides were injected subcutaneously and examined for various immunological indicators. The results show that when gangliosides were administered in the effector phase, TMEV-IDD was suppressed both clinically and histologically. Cellular immunity such as delayed-type hypersensitivity, and the proliferative response of T cells against TMEV and mitogens were decreased, and only in this group anti-TMEV IgG2a antibody was not detected. Taken together, these data suggest that administration of gangliosides suppressed the function of pathogenic Th1 cells and suppressed TMEV-IDD. Additionally, this study proposes the possibility of a new therapy in multiple sclerosis.

Headache associated with aseptic meningeal reaction as clinical onset of Fabry's disease.

This report concerns an 18-year-old boy who is hemizygote for Fabry's disease. Varying degrees of nonpulsating headache crises, lasting from a few hours to several days, began when he was 16 years of age. Painful crises in the extremities, characteristic of Fabry's disease, were not present. Although only occasional, he had several episodes of throbbing headache with vomiting without aura. The meningeal signs were equivocal, although the patient had noninfectious pleocytosis, intracranial hypertension, delayed radioisotope clearance on cisternography, and multiple old cerebral infarcts. Nonsteroidal anti-inflammatory drugs, antidepressants, carbamazepine, and glycerol were of no benefit for his headache. Although its mode of action remains obscure, prednisolone was effective for treating the headache and the aseptic meningeal reaction.