Regulation of synaptic strength by sphingosine 1-phosphate in the hippocampus.

Although the hippocampus is a brain region involved in short-term memory, the molecular mechanisms underlying memory formation are not completely understood. Here we show that sphingosine 1-phosphate (S1P) plays a pivotal role in the formation of memory. Addition of S1P to rat hippocampal slices increased the rate of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) recorded from the CA3 region of the hippocampus. In addition long-term potentiation (LTP) observed in the CA3 region was potently inhibited by a sphingosine kinase (SphK) inhibitor and this inhibition was fully reversed by S1P. LTP was impaired in hippocampal slices specifically in the CA3 region obtained from SphK1-knockout mice, which correlates well with the poor performance of these animals in the Morris water maze test. These results strongly suggest that SphK/S1P receptor signaling plays an important role in excitatory synaptic transmission in the CA3 region of hippocampus and has profound effects on hippocampal function such as spatial learning.

Intra-articular oxidative state correlated with the pathogenesis of disorders of the temporomandibular joint.

We investigated the redox state of albumin in the synovial fluid from patients with temporomandibular joint (TMJ) disorders (TMD) to evaluate the relation between the cause of the TMD and the number of types of oxygen in synovial fluid. The albumin was fractionated into three components, human mercaptalbumin (HMA, reduced form) and two types human non-mercaptalbumin (HNA, oxidized form), by high-performance liquid chromatography (HPLC). The 63 patients were divided into three groups radiologically, and the ratios of the redox state of the synovial fluid in each group were compared. The fraction of HNA was significantly higher in patients with advanced disease than in patients with early disease. This indicates that the TMJ is affected by intra-articlular oxidative stress, and the severity of TMD correlates closely with the number of oxidative factors. Oxidative stress was thought to be responsible for the genesis of TMD.

Application of L-threonine aldolase-catalyzed reaction to the preparation of protected 3R,5R-dihydroxy-L-homoproline as a mimetic of idulonic acid.

A facile synthesis of 3R,5R-dihydroxy-L-homoproline as idulonic acid mimic, of which the carboxyl and 3-hydroxyl groups were protected, was attained using L-threonine aldolase-catalyzed reaction. Idulonic acid is a key acidic sugar of the b-FGF binding domain in heparin and heparan sulfate. Moreover, the synthetic precursor of N-acetyl-4-deoxy-D-mannosamine, which is a potent inhibitor of NeuAc synthase, was prepared from the side product of the enzymatic aldol condensation.

Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells.

We investigated the functional roles of ceramide, an intracellular lipid mediator, in cell signaling pathways by monitoring the intracellular movement of protein kinase C (PKC) subtypes fused to green fluorescent protein (GFP) in HeLa living cells. C(2)-ceramide but not C(2)-dihydroceramide induced translocation of delta PKC-GFP to the Golgi complex, while alpha PKC- and zeta PKC-GFP did not respond to ceramide. The Golgi-associated delta PKC-GFP induced by ceramide was further translocated to the plasma membrane by phorbol ester treatment. Ceramide itself accumulated to the Golgi complex where delta PKC was translocated by ceramide. Gamma interferon also induced the delta PKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and Mg(2+)-dependent neutral sphingomyelinase. Photobleaching studies showed that ceramide does not evoke tight binding of delta PKC-GFP to the Golgi complex but induces the continuous association and dissociation of delta PKC with the Golgi complex. Ceramide inhibited the kinase activity of delta PKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of delta PKC-GFP immunoprecipitated from ceramide-treated cells was increased. The immunoprecipitated delta PKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosine kinase inhibitor abolished the ceramide-induced activation and tyrosine phosphorylation of delta PKC-GFP. These results suggested that gamma interferon stimulation followed by ceramide generation through Mg(2+)-dependent sphingomyelinase induced delta PKC-specific translocation to the Golgi complex and that translocation results in delta PKC activation through tyrosine phosphorylation of the enzyme.

Ganglioside GD3 and its mimetics induce cytochrome c release from mitochondria.

Ganglioside GD3 induced the release of cytochrome c from isolated rat liver mitochondria. This process was completely prevented by cyclosporin A and partially prevented by a cysteine protease inhibitor, n-acetyl-leu-leu-norleucinal. Cyclosporin A is a potent inhibitor of the permeability transition pore, whereas n-acetyl-leu-leu-norleucinal has no effect on this pore. These results indicate that the release of cytochrome c from mitochondria requires both the opening of the permeability transition pore and a cysteine protease inhibitor-sensitive mechanism. Gangliosides GD1a, GD1b, GT1b, and GQ1b along with the synthetic GD3 mimetics TMS-42 and CI-22, which are glycerophospholipids carrying a disialo residue, also induced cytochrome c release. In contrast, gangliosides GM1, GM2, and GM3 did not induce cytochrome c release. These results indicate that two sialo residues must play an important role in the induction of cytochrome c release by gangliosides.

Neurogenic pathways mediating ascending and descending reflexes at the porcine ileocolonic junction.

We studied the pharmacology of the neural pathways mediating the responses of ileo- and coloileo-colonic junction (ICJ) to regional distension in ten anaesthetized pigs. Using manometric pullthroughs and a sleeve sensor, we found the ICJ demonstrated sustained tone that was resistant to tetrodotoxin. Ileal distension decreased ICJ pressure by 22.2 ¿ 10.1% (11.9 ¿ 2.7-10.1 ¿ 2.6 mmHg; P=0.002) and colonic distension augmented ICJ pressure by 23.5 ¿ 8.6% (12.8 ¿ 1.5-15.6 ¿ 2.1 mmHg; P=0.02). Bethanecol and Nw-nitro-L-arginine methyl ester (L-NAME) increased ICJ pressure (P=0.002, P=0.01, respectively). Sodium nitroprusside and isoproterenol reduced ICJ pressure (P=0.004, P=0.02, respectively). In the presence of L-NAME, the early inhibitory ileo-ICJ response was abolished, while early and late inhibitory responses were abolished by further addition of propranolol but not by the addition of hexamethonium, atropine, prazosin or yohimbine. The excitatory colo-ICJ response was replaced by inhibition in the presence of L-NAME. We concluded that: (1) the porcine ICJ displays myogenic tone which is influenced by excitatory muscarinic and inhibitory nitrergic and beta adrenergic pathways (2) an inhibitory ileo-sphincteric reflex mediated by nitrergic and beta adrenergic postganglionic neural pathways (3) both excitatory and inhibitory neurogenic colo-sphincteric reflexes exist, and the excitatory pathway involves nitrergic neurotransmission.

A 66-base-pair enhancer module activates the expression of a distinct isoform of UDP-glucuronosyltransferase family 1 (UGT1A2) in primary hepatocytes.

UGT1A2, an isoform of the UDP-glucuronosyltransferase family 1 (UGT1), is not expressed in the rat liver, but its expression was highly induced in primary cultures of rat hepatocytes. In primary hepatocytes that had been cultured for 70 h, the amount of UGT1A2 mRNA was 100 times higher than that in the rat liver. Deletion analysis of a 4.8-kb promoter region of the UGT1A2 gene revealed that a 66-nucleotide region between -307 and -242 upstream of the transcription start site was required for induction of UGT1A2 expression. The 66-nucleotide region acted on a heterologous promoter in a manner independent of its position and orientation in reporter constructs. Gel mobility shift assay showed that a specific binding protein to this region appeared in the nuclei of cultured hepatocytes, but was not present in the rat liver. DNase I protection analysis revealed the existence of a CTGGCAC core sequence between -274 and -268 of the UGT1A2 promoter. Methylation interference assay showed that the guanine residues at -294 and -287 on the upper strand and the guanine residue at -267 on the lower strand as well as the core sequence were required for the DNA-protein interaction. These results suggest that the 66-nucleotide region, which was designated culture-associated expression responsive enhancer module (CEREM), interacts with a specific nuclear protein and enhances the expression of UGT1A2 in cultured hepatocytes.

[Synthesis of carbohydrate related compounds by using aldolase catalyzed reaction].

Enzymes proceed the reaction with high regio- and stereoselectivity under mild conditions, i.e. in an aqueous medium at room temperature. However, enzymatic reactions that catalyze carbon-carbon bond formation have not been utilized in organic synthesis until recently. We had an interest in an aldolase-catalyzed reaction which proceed carbon-carbon bond formation referred to aldol condensation, by which many bioactive compounds have been rationally synthesized. On the other hand, recent biological studies on cell recognition (cell adhesion) have disclosed the important roles of oligosaccharides on cell surfaces, especially which include glucuronic acid, 3-deoxy-D-manno-oct-2-ulosonic acid (KDO), and sialic acid in the structures e.g., sialyl Lewis X and endotoxins, in differentiation, induction, viral and bacterial infections, and immune response. As well as acidic oligosaccharides, basic ones have been utilized as practical medicines in the clinical level, like acarbose that acts as an amylase inhibitor. Based on these background, we embarked the synthesis of carbohydrate related compounds which can control the interaction between carbohydrates and carbohydrate recognition protein by the use of several aldolases. Azasugars, potent inhibitors toward glycosidases, were synthesized using fructose-1,6-diphosphate (FDP)-aldolase and other dihdroxyacetonephosphate (DHAP)-dependent aldolases in the key step. Sialyl Lewis X mimetic, peptidic mimetic of RNA having anti-Vero toxin activity, mycestericin D, and aza-idulonic acid were prepared by taking advantage of L-threonine aldolase catalyzed reaction, which afford beta-hydroxy-alpha-L-amino acids. A precursor of KDO, featured acidic sugar of endotoxins was provided by the reaction catalyzed with kynureninase, which generates beta-anion of L-alanine in its active site during the metabolic reaction from kynurenine to anthranilic acid.

Above- and belowground biomass and primary productivity of a Larix gmelinii stand near Tura, central Siberia.

We assessed above- and belowground biomass and net primary production (NPP) of a mature Larix gmelinii (Rupr.) Rupr. forest (240-280 years old) established on permafrost soils in central Siberia. Specifically, we investigated annual carbon budgets in roots in relation to root system development and availability of soil resources. Total stand biomass estimated by allometry was about 39 Mg per ha. Root biomass (17 Mg per ha) comprised about 43% of total biomass. Coarse root (>/= 5 mm in diameter) biomass was about twice that of fine roots (< 5 mm). The aboveground biomass/root biomass ratio (T/R) of the larch stand was about unity, which is much less than that of other boreal and subalpine conifer forests. The proportion of fine roots in total root biomass (35%) was relatively high compared with other cold-climate evergreen conifer forests. Total NPP, defined as the sum of annual biomass increment of woody parts and needle biomass, was estimated to be 1.8 Mg per ha per year. Allocation of total NPP to needle production was 56%. The proportion of total NPP in belowground production (27%) was less than for evergreen taiga forests. However, belowground NPP was probably under-estimated because root mortality was excluded. We conclude that L. gmelinii trees invested annual carbon gains largely into needle production or roots, or both, at the expense of growth of aboveground woody parts. This carbon allocation pattern, which resulted in the construction of exploitative root networks, appeared to be a positive growth response to the nutrient-poor permafrost soil of central Siberia.

Feline interferon-omega treatment on canine parvovirus infection.

Recombinant feline interferon-omega preparation (rFeIFN-omega, trade name: INTERCAT) showed good clinical efficacy on canine parvovirus infection both in an experimental trial with beagles, and in field trials.

Basal pressure patterns and reflexive motor responses in the human ileocolonic junction.

This study aimed to determine whether a sustained high-pressure zone exists at the human ileocolonic junction (ICJ) and whether the motor responses of ICJ are consistent with sphincteric function. In 10 subjects with temporary ileostomies, a high-pressure zone was identified using a manometric pull-through with a mean pressure of 9. 7 +/- 3.2 mmHg and length of 4.8 +/- 1.2 cm. Prolonged recordings using a sleeve sensor confirmed sustained tone in the ICJ and superimposed phasic pressure waves (4-8 counts/min) occupying 35% of fasted state. A meal increased ICJ tone (P = 0.0001) and the proportion of time occupied by phasic activity to 50% (P = 0.013). Terminal ileal propagating pressure wave sequences inhibited ICJ phasic activity, and sequences not extending to the cecum reduced ICJ tone (9.0 +/- 7.2 to 5.6 +/- 6.3 mmHg; P = 0.04). Cecal distension increased ICJ tone (8.9 +/- 4.4 mmHg to 11.7 +/- 4.9 mmHg; P = 0.005). The ICJ response to ileal distension was variable and depended on resting tone at the time of distension. We conclude that the human ICJ has sustained tone with superimposed phasic activity. Tone is augmented by cecal distension or a meal and is inhibited by ileal propagating pressure waves. Response to ileal distension is variable but suggests control by descending excitatory and inhibitory pathways.

Synthesis and evaluation of morpholino- and pyrrolidinosphingolipids as inhibitors of glucosylceramide synthase.

This paper describes the new synthesis and evaluation of some morpholino- and pyrrolidinosphingolipids and mimics as inhibitors of glucosylceramide synthase. It was found that the pyrrolidino derivatives are generally more active than the morpholino derivatives and the best one was shown to be a nanomolar inhibitor.

Clinical effects of the recombinant feline interferon-omega on experimental parvovirus infection in beagle dogs.

The clinical effects of recombinant feline interferon-omega (rFeIFN-omega), produced in silkworm by recombinant baculovirus, were examined in 3-4 month-old beagle dogs given an experimental canine parvovirus type-2 (CPV-2) infection. Clinical symptoms, such as pyrexia, vomiting, anorexia and diarrhea, were observed on day 4 after oral inoculation of 10(7) TCID50 of CPV-2 (cc 238 strain) in almost all the inoculated dogs. From day 4, rFeIFN-omega (1 mega units/kg/day) or physiological saline was administered intravenously to infected dogs for 3 consecutive days. Seven out of 17 dogs treated with physiological saline showed hemorrhagic diarrhea and continuously expressed severe clinical enteritis; one dog died with a large amount of hemorrhagic rice-water stool on day 6 after viral exposure. In contrast, 4 out of 12 dogs treated with rFeIFN-omega showed severe clinical enteritis associated with intermittent diarrhea. Scoring of fecal condition revealed that treatment with rFeIFN-omega significantly shifted the enteritis from a severe to mild form. Furthermore, rFeIFN-omega administered in the morning decreased the number of dogs expressing clinical enteritis in the evening suggesting a rapid effect. Vomiting and anorexia were also improved by treatment with rFeIFN-omega. These results suggest that rFeIFN-omega can reduce severe enteritis caused by CPV-2 infection in dogs.

Metabolic labeling of a subset of glial cells by UDP-galactose: implication for astrocyte lineage diversity.

Astrocytes are implicated in many aspects of brain function; however, it remains unclear whether astrocytes arise from a single cell lineage. It is therefore important to obtain new markers for the astrocyte cell lineage. We show that exogenously added UDP-galactose (UDP-Gal) can be used to metabolically label a subset of glial fibrillary acidic protein-positive (GFAP+) cells. UDP-Gal was incorporated into the cultured embryonic mouse brain slices in a time-dependent manner. Surprisingly, the transferred sugar moiety was no longer Gal but was mainly glucose. Most of the radioactivity was transferred to a polymer of glucose, most likely to be glycogen, and also to glucosyl ceramide. In the slice culture, the reaction products were distributed densely in the ventricular zone and also on process-like structures extending to the pial surface. In dissociation culture, UDP-Gal labeled some of the GFAP+ cells and some of the vimentin+ cells. Because radial glial cells (RGCs) contain glycogen and change from vimentin+ to GFAP+, it is strongly suggested that UDP-Gal labeled RGCs and their descendants. Only 27% of the GFAP+ cells were labeled with UDP-Gal, which suggests that only a subset of astrocytes are derived from RGCs and that there is a discrete group of GFAP+ cells that is not generated from RGCs.

Sintered carbonate apatites as bioresorbable bone substitutes.

The dissolution behavior of sintered carbonate apatite was investigated in a 10 mM/L acetic acid solution adjusted to pH 5.0 at 37 degrees C, and compared to that of sintered hydroxyapatite and bone apatite for the purpose of establishing some similarities between the physicochemical dissolution of apatite biomaterials in vitro and their ability to be resorbed by osteoclasts in vivo. Both the sintered carbonate apatite and the bone apatite dissolved to an appreciable extent. Their solution compositions changed in an almost identical manner until toward the end of the reaction. The solution compositions for sintered carbonate apatite at 30 s was comparable with that for sintered hydroxyapatite at 3.8 days with respect to the degree of supersaturation, indicating that the former specimen is much more soluble than the latter specimen. Osteoclasts which were obtained from the long bones of 1-day-old neonatal rabbits resorbed bone and sintered carbonate apatite, but not sintered hydroxyapatite. These findings suggest that sintered carbonate apatites, which have characteristics that can be favorably compared with those of bone, especially with respect to its reactivity to acid media, would be useful as bioresorbable bone substitutes.

Synthetic sialylphosphatidylethanolamine derivatives bind to human influenza A viruses and inhibit viral infection.

We synthesized the sialylphosphatidylethanolamine (sialyl PE) derivatives Neu5Ac-PE, (Neu5Ac)2-PE, Neu5Ac-PE (amide) and Neu5Ac-PE (methyl). We examined the anti-viral effects of the derivatives on human influenza A virus infection by ELISA/virus-binding, hemagglutination inhibition, hemolysis inhibition and neutralization assays. The sialyl PE derivatives that we examined bound to A/Aichi/2/68, A/Singapore/1/57 and A/Memphis/1/71 strains of H3N2 subtype, but not to A/PR/8/34 strain of H1N1 subtype. The derivatives inhibited viral hemagglutination and hemolysis of human erythrocytes with A/Aichi/2/68 and A/Singapore/1/57 (H3N2), but not with A/PR/8/34 (H1N1). The inhibitory activity of the (Neu5Ac)2-PE derivative was the strongest of all sialyl PE derivatives (IC50, 35 microM to 40 microM). Sialyl PE derivatives also inhibited the infection of A/Aichi/2/68 in MDCK cells. Complete inhibition was observed at a concentration between 0.3 to 1.3 mM. IC50 of (Neu5Ac)2-PE was 15 microM in A/Aichi/2/68 strain. Taken together, the synthetic sialyl PE derivatives may be effective reagents against infection of some types of influenza A viruses.

Requirement of the glycosyl parts in platycodin D to stimulate pancreatic exocrine secretion.

The whole structure of platycodin D is found to be essential to stimulate the volumetric increase in the pancreatic exocrine secretion, while the prosapogenins prepared from platycodin D increased only protein output of pancreatic juice.

Two novel oleanolic acid saponins having a sialyl Lewis X mimetic structure from Achyranthes fauriei root.

Two novel triterpene glycosides, achyranthosides E and F, were isolated as methyl esters from the root of Achyranthes fauriei, an antiinflammatory medicinal plant. Their structures were characterized as oleanolic acid glucuronides having unique substituents composed of C6H9O5 and C9H15O7, respectively, at the C-3 position of glucuronic acid. These compounds are active components which can inhibit the excess recruiting of neutrophiles to injured tissues 1,000 times more potently than sialyl Lewis X.

Synthesis of a novel sialic acid derivative (sialylphospholipid) as an antirotaviral agent.

A novel sialylphospholipid (SPL) was synthesized from N-acetylneuraminic acid (NeuAc) and phosphatidylcholine (PC) by a chemical and enzymatic method and evaluated as an inhibitor of rotavirus. PC and 1,8-octanediol were conjugated by transesterification reaction of Streptomyces phospholipase D (PLD) under a water-chloroform biphasic system to afford phosphatidyloctanol, which was condensed with a protected 2-chloro-2-deoxyneuraminic acid derivative by using silver trifluoromethanesulfonate as an activator in chloroform and converted, after deprotection, to SPL. Rhesus monkey kidney cells (MA-104) were incubated with simian (SA-11 strain) and human (MO strain) rotaviruses in the presence of SPL, and the cells infected were detected indirectly with anti-rotavirus antibody. SPL showed dose dependent inhibition against both virus strains. The concentrations required for 50% inhibition (IC50) against SA-11 and MO were 4.35 and 16.1 microM, respectively, corresponding to 10(3)- and 10(4)-fold increases in inhibition as compared to monomeric NeuAc.

A search for pyrophosphate mimics for the development of substrates and inhibitors of glycosyltransferases.

The design and synthesis of several beta-1,4-galactosyltransferase inhibitors are reported. Mimics of the pyrophosphate-Mn2+ complex were the focus of the design. Malonic, tartaric, and monosaccharide moieties were used as replacements of the pyrophosphate moiety, and galactose or azasugars with potent galactosidase inhibitory activity were used as the 'donor' component. Compound 6, in which glucose was used as the pyrophosphate-Mn2+ complex mimic and galactose as the 'donor' component, showed the best inhibitory activity towards the transferase with a Ki of 119.6 microM.