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.

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.

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.

Steroidogenic effect of ent-kaur-16-en-15 beta-ol (kaurenol) on isolated rat adrenal cells.

In an in vitro bioassay system for adrenocorticotropic hormone using isolated rat adrenal cells, kaurenol, a diterpene alcohol, stimulated corticosterone production and augmented the steroidogenic effect of adrenocorticotropin or forskolin, dose-dependently. Kaurenol had no effect on cyclic AMP production by the cells. The diterpene also had no stimulatory effect on the adrenal adenylate cyclase activity in a cell free system. The results suggest that this particular diterpene exerts a steroidogenic effect through a mechanism independent of cyclic AMP generation.

[Relation between electrogastrography and gastric electromyogram, mechanical activity].

Electrogastrography (EGG) appears to be useful method to evaluate the gastric motility as noninvasive approach. However, it is not known what EGG means. The aim of this study is to make clear the meanings of electrical activity recorded dy EGG for gastric motility. EGG recorded by cutaneous electrodes was compared to gastric electromyogram recorded by serosal electrodes and gastric mechanical activity recorded by straingauge transducer in anesthetized and conscious dogs. EGG recordings were preserved when gastric mechanical activity was not recognized. In digestive state, amplitude of EGG, electromyogram and mechanical activity increased significantly (p < 0.01). Significant correlation between amplitude of EGG and amplitude of electromyogram and mechanical activity of antrum were seen (p < 0.01). In interdigestive state, amplitude of EGG in phase 3 increased significantly (p < 0.05) compared to that in phase 1. Significant correlation between amplitude of EGG and amplitude of electromyogram and mechanical activity were seen in phase 2 (p < 0.01). EGG reflected electrical control activity (ECA) and electrical response activity (ERA). EGG recordings showed electromyogram and mechanical activity of gastric antrum. Amplitude of EGG reflected electromyogram and mechanical activity in phase 2 in interdigestive state. EGG waves did not always agree on myoelectrical waves.

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.

Hepatocellular carcinoma complicating biliary cirrhosis caused by biliary atresia: report of a case.

A case of hepatocellular carcinoma complicating biliary cirrhosis caused by biliary atresia is reported. The patient had persistent severe jaundice with hepatosplenomegaly. A liver tumor was suspected because of the elevated serum alpha-fetoprotein and was shown by ultrasonography at 6 years of age. The tumor was treated with percutaneous ethanol injection therapy (PEIT). Nine months after initiation of PEIT, the patient died of massive bleeding from a metastatic tumor.

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.

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.

[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.

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.

Shoot dynamics of Pinus pumila in relation to altitudinal and wind exposure gradients on the Kiso mountain range, central Japan.

Seasonal shoot dynamics of Japanese mountain stone pine (Pinus pumila Regel.) growing at six sites with different altitudes and slope aspects on the Kiso mountain range in central Japan were investigated. The shoots followed a similar growth pattern at all sites; current shoots elongated rapidly during June and July, and the current needles grew during July and August. Final lengths of the current shoots and needles decreased with increasing altitude. Current-shoot lengths were also shorter at the windward sites than at the leeward sites. At all sites, leaf senescence occurred intensively between mid-August and mid-September. Needle longevity, i.e., the age of the oldest living needle attached to a shoot, increased with increasing altitude. Fascicle density, i.e., the number of current needle fascicles on a unit shoot length, was greater at higher altitudes and windward sites than at lower altitudes and leeward sites. Seasonal dynamics of the P. pumila shoots were characterized by simultaneous replacement of old needles with new needles in the early autumn, thus avoiding any loss of canopy photosynthetic production during the growing season. Increases in needle longevity and fascicle density were associated with declining air temperature and increasing wind exposure. Needle longevity and fascicle density were characteristics of adaptive plasticity in P. pumila that prevent a reduction in growth potential in the stressful conditions of alpine regions.

[Basic study of the application of 2 piece-type hydroxyapatite anchor in periodontal therapy].

Clinico-histological studies were made to obtain basic data on the application of 2 piece-type hydroxyapatite (hereinafter referred to as HAP) originally designed and prepared so as to be shaped to make its clinical application easy as an anchor (HAP anchor) in the corrective movement of teeth. An immobility identification experiment and intrusive experiment were performed in order to identify absent loss of anchor to the corrective force and to identify actually practicable tooth movement by means of the HAP anchor, respectively, and clinico-histological evaluation was made of its function in both of the above cases. The results revealed no change in the distance of HAP anchor in the former experiment and tooth intrusion of 3.7 mm on average in the latter experiment and, histologically, close surrounding of almost the entire region embedded by the HAP anchor, with newly formed osseous tissues in both experiments. The above results suggest the usefulness of this trial HAP anchor as an anchor to tooth movement in periodontal therapy.

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.

The internal anal sphincter in anorectal malformation.

Recently it has been morphologically clarified that a layer of thick smooth muscle, mimicking the internal anal sphincter, exists at the rectal and even in high and intermediate anorectal malformations. This paper will describe two studies focussed on the internal anal sphincter in anorectal malformation. I. Experimental study: Utilisation of the rectal end for reconstructive surgery would necessitate the mobilisation of the rectum. We performed a chronic animal experiment in order to investigate the influence of rectal mobilisation on anal function and the role of the internal anal sphincter in anal continence. Fifteen dogs were operated on; rectal separation was performed in 5 dogs, resection of the internal anal sphincter in 5 dogs and both procedures in 5 dogs. Anorectal manometric studies for 24 weeks revealed that rectal separation caused only a transient mild disturbance in anal functions with the exception of long-standing high rectal compliance, while resection of the internal anal sphincter caused a persistent severe disturbance. II. Clinical study: The function of the smooth muscle thickening at the rectal end was investigated and operations to preserve the rectal end were evaluated in cases of high and intermediate anorectal malformations. In 5 infants with anorectal malformations (high type 2, intermediate type 3), for whom colostomies had been performed as newborn, a preoperative manometric study at the rectal end was performed with a probe introduced from the distal colostomy. Thereafter, they all underwent a rectal end preserving operation. They were followed up manometrically and clinically after the operation. A preoperative manometric study of the rectal end showed the presence of rhythmic activity in all and positive reflexive pressure fall by rectal distension in 4. The rectal end preserving operation maintained rhythmic activity in all of them, rectoanal reflex in 3 and good results are expected clinically. These two studies suggest that the functional internal anal sphincter, which exists at the rectal end also in high and intermediate anorectal malformations, should be utilised for reconstruction because of the essential role it plays in anal continence.

Nuclear magnetic resonance studies of cytochalasin E and its decomposition product.

The proton and carbon-13 nuclear magnetic resonance signals of cytochalasin E (1) were assigned with the aid of 1H-1H, 1H-13C and 1H-13C long-range chemical shift correlation spectroscopy spectra, and the structure of the decomposition product (2) generated under neutral conditions was determined.

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.