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Biomedical subjects

H Fukuda

Publications and source records attributed to H Fukuda.

At least 19 recordsLinked to original sources

Two reactions are simultaneously catalyzed by a single enzyme: the arginine-dependent simultaneous formation of two products, ethylene and succinate, from 2-oxoglutarate by an enzyme from Pseudomonas syringae.

A single enzyme isolated from Pseudomonas syringae pv. phaseolicola PK2 simultaneously catalyzed two reactions, namely, the formation of ethylene and succinate from 2-oxoglutarate, at a molar ratio of 2:1. In the main reaction, 2-oxoglutarate was dioxygenated to produce one molecule of ethylene and three molecules of carbon dioxide. In the sub-reaction, both 2-oxoglutarate and L-arginine were mono-oxygenated to yield succinate plus carbon dioxide and L-hydroxyarginine, respectively, the latter being further transformed to guanidine and L-delta 1-pyrroline-5-carboxylate. We propose a dual-circuit mechanism for the entire reaction, in which the binding of L-arginine and 2-oxoglutarate in a Schiff-base structure generates a common intermediate for two reactions.

Arginine

Identification of the binding site of 55kDa tumor necrosis factor receptor by synthetic peptides.

We have synthesized a series of peptides, which cover almost the whole range of the N-terminal extracellular domain of human 55kDa TNF receptor (55kDa TNF-R). The peptides were examined for the binding activity to TNF by solid phase binding assay and for the inhibition of TNF cytotoxicity to mouse L-M cells. The peptide 159-178 exhibited remarkably higher binding activity to TNF than other peptides did. The specificity of the TNF binding to the peptides was confirmed by their inability to bind other cytokines. The peptide 159-178 also inhibited TNF cytotoxicity. These results indicate that the specific binding site of 55kDa TNF-R to TNF might reside within the peptide segment of amino acid numbers 159 to 178 in the N-terminal extracellular domain.

Amino Acid Sequence

Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2.

The gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2 was found to be encoded by an indigenous plasmid, designated pPSP1. The gene for the ethylene-forming enzyme was cloned and expressed in Escherichia coli JM109. Nucleotide sequence analysis of the clone revealed an open reading frame that encodes 350 amino acids (mol. wt. 39,444). In a comparison with other proteins, the homology score for the entire amino-acid sequence of the ethylene-forming enzyme of Pseudomonas syringae versus ethylene-forming enzymes from plants and 2-oxoglutarate-dependent dioxygenases was low. However, functionally significant regions are conserved.

Amino Acid Sequence

Archaebacterial lipid models: highly salt-tolerant membranes from 1,2-diphytanylglycero-3-phosphocholine.

1,2-Di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine and its glycerol epimers were synthesized as model lipids of archaebacterial halophiles. These amphiphiles, upon sonication of aqueous suspensions, gave rise to small unilamellar vesicles (SUV) of 300-800 A in diameter and about 80 A in the membrane thickness. The liposomes were very stable for at least a month even in a highly concentrated suspension or 5 M aqueous NaCl. The vesicles could retain Na+ and Cl- ions as well as 5(6)-carboxyfluorescein in the aqueous interior at temperature as high as 70 degrees C. The liposomes of ordinary diester lipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and egg-yolk lecithin were less stable and more permeable than those of the diphytanyl lipids.

Archaea

A gene expressed preferentially in the globular stage of somatic embryogenesis encodes elongation-factor 1 alpha in carrot.

We have isolated cDNA of genes that are preferentially expressed during somatic embryogenesis of carrot (Daucus carota L.) by differential screening of globular embryos and cells that are dividing in an unorganized manner. As a result of Northern-blot analysis, one of the genes identified in this way, which we refer to as CEM1, was found to be expressed at high levels in somatic embryos at the globular and heart-shaped stages. In-situ hybridization using globular embryos revealed that the mRNA transcribed from CEM1 was located preferentially in the spherical region of the globular embryo. A homology search using the amino acid sequence deduced from the nucleotide sequence of the CEM1 cDNA revealed that CEM1 encodes the eukaryotic translational elongation-factor 1 alpha.

Amino Acid Sequence

Effects of nutrients and hormones on gene expression of ATP citrate-lyase in rat liver.

Northern-blot analyses demonstrated a strong gene expression of ATp citrate-lyase in liver and adipose tissue of rat and a weak expression in brain, heart, small intestine and muscle. After refeeding a carbohydrate/protein diet to fasted rats, the transcriptional rate had already increased within 2 h, the mRNA concentration reached a maximal level of approximately 30-fold increased in 16 h, and the enzyme induction increased sixfold in 48 h. By feeding only carbohydrate without protein, the transcriptional rate was increased threefold, and the mRNA concentration and enzyme induction comparably, to the levels in the carbohydrate/protein diet. It appears that protein feeding is not necessary to induce ATP citrate-lyase. In diabetic rats fed on a glucose diet, the transcriptional rate, mRNA concentration and enzyme level were very low in comparison with the normal. By fructose feeding, however, the transcriptional rate was more greatly increased and the mRNA concentration increased comparably to the levels reached by insulin treatment, while the enzyme induction was not so increased. Thus, it is suggested that insulin is important in regulated translation in addition to transcription. However, triiodothyronine treatment did not have much effect on the gene expression. As a result of the present experiment, it is noted that ATP citrate-lyase-gene expression was greatly dependent on carbohydrate.

ATP Citrate (pro-S)-Lyase

5-HT2/5-HT1C receptor-mediated facilitatory action on unit activity of ventral horn cells in rat spinal cord slices.

5-Methoxy-N,N-dimethyltryptamine (5-MeODMT) and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) facilitate motoneuron excitability through 5-HT1C/5-HT2 receptors in rats. Using spinal cord slices prepared from adult rats, we recorded unitary cell discharges, evoked by local stimulation of the adjacent site, extracellularly in the motor nuclei of the ventral horn. 5-MeODMT, DOI, 5-hydroxytryptamine (5-HT), 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) and tandospirone facilitated the probability of firing in the motor nuclei, with 5-MeODMT and DOI being the most potent. The effect of 5-MeODMT was significantly suppressed by ketanserin (a 5-HT2 receptor-selective antagonist), spiperone (a 5-HT1A/5-HT2 receptor antagonist) and cyproheptadine (a 5-HT1C/5-HT2 receptor antagonist), but not by 3-tropanyl-3,5-dichlorobenzoate (MDL 72222, a 5-HT3 receptor-selective antagonist) or pindolol (a 5-HT1A/5-HT1B receptor antagonist). This suggests that 5-HT2 and/or 5-HT1C receptors are involved in the facilitatory effects of 5-HT receptor agonists on the synaptic activity of ventral horn cells.

8-Hydroxy-2-(di-n-propylamino)tetralin

[Gd-DTPA-enhanced MR imaging of the normal facial nerve].

We performed a prospective imaging study of the normal facial nerve within the temporal bone before and after injection of Gd-DTPA. The study included 29 patients using a 1.5T superconducting unit and 40 nerves (right: 21; left: 19) were available for analysis. There was no enhancement of the facial nerve within the internal auditory canal in the entire series. However, the enhancement at the labyrinthine segment was observed in one nerve (3%); at the geniculate ganglion in seven (18%); at the tympanic segment in 18 (45%) and at the mastoid segment in 28 nerves (70%). Our results indicate that enhancement of the facial nerve in normal subjects is not a rare condition.

Adolescent

Stimulation of three areas of the primary motor cortex interrupts micturition in dogs.

To clarify the area of the motor cortex (M1) in dogs, which corresponds to the cortical area participating in voluntary interruption of micturition in humans, the cortical portions related to the external urethral sphincter were first clarified by recording of somato-sensory evoked potentials, and then systematic cortical stimulation was performed in anesthetized and paralyzed dogs. The hypogastric, pelvic and pudendal nerves innervating the lower urinary tract and rectum were severed to eliminate the secondary reflexes. Five foci were recognized in the cortical potentials evoked after stimulation of the pudendal nerve. These foci existed in the sacral (Sacral-S) and hind leg (Leg-S) areas of the somato-sensory cortex (S1), and in the sacral (Sacral-M), hind leg (Leg-M) and trunk (Abd-M) areas of the M1. Stimulation of the three M1 foci, but not the two S1 foci, provoked centrifugal firings of the pudendal urethral branch. The firings disappeared after cutting of the ipsilateral bulbar pyramis. When the M1 foci were stimulated during the micturition reflex, the reflex discharge of a pelvic vesical branch was interrupted concomitantly with firings of the urethral branch. The interruption was still induced after the pyramidotomy. Pulse train stimulation of these M1 foci reset the cycles (about 2 Hz) in the alternative rhythmic firings of the urethral and vesical branches, which are known to be formed in the pontine micturition center [31,32]. These results suggest that the pyramidal cells in the three M1 foci inhibit the pontine micturition center and concomitantly contract the external urethral sphincter through the pyramidal tract. The possible roles of these M1 foci were discussed.

Animals

Stimulatory effects of islet amyloid polypeptide (amylin) on exocrine pancreas and gastrin release in conscious rats.

A rat islet amyloid polypeptide (amylin), 37-residue peptide amide was synthesized by the Fmoc-based solid phase method and the biological activity of synthetic rat amylin on exocrine pancreas was evaluated for the first time in conscious rat. Amylin (1, 10 nmol/kg/h) stimulated pancreatic exocrine secretion and plasma gastrin concentration. CR-1409, a CCK receptor antagonist, did not change amylin-stimulated pancreatic secretion. However, omeprazole (proton pump inhibitor) and atropine inhibited amylin-stimulated pancreatic secretion. This study suggests that amylin may play a role in biological action in the exocrine pancreas possibly mediated by gastric acid hypersecretion.

Amyloid

Cyclic AMP analog activates Na(+)-dependent inward currents in dissociated frog motoneurons.

Effects of intracellular accumulation of 3',5'-cyclic adenosine monophosphate (cAMP) were studied using 3',5'-cyclic 8-bromoadenosine monophosphate (8-Br-cAMP) and forskolin on single motoneurons acutely dissociated from adult bullfrog spinal cord. 8-Br-cAMP (10(-3) M) and forskolin (1.5 x 10(-6) M) activated inward currents under K(+)-free conditions at a holding potential of -70 mV. The currents were dependent on extracellular Na+ concentration, and were never reversed within the range of membrane potentials tested (-130 to 30 mV). These results indicate that accumulation of intracellular cAMP induces Na(+)-dependent inward currents in frog motoneurons.

8-Bromo Cyclic Adenosine Monophosphate

Activation of beta-adrenergic receptor induces Na(+)-dependent inward currents in acutely dissociated motoneurons of bullfrog spinal cord.

Effects of adrenergic drugs on single motoneurons acutely dissociated from the lumbar enlargement of adult bullfrogs were examined. The dissociated large cells were identified as motoneurons by retrograde labeling with a fluorescent dye. Adrenaline caused membrane depolarization with a decrease in input resistance. Under whole-cell voltage clamp conditions at a holding potential of -70 mV, adrenergic drugs induced inward currents in a dose-dependent manner. Adrenaline was more potent than noradrenaline. Under K(+)-free conditions, adrenaline (10(-6)-10(-5) M) induced inward currents which were blocked by propranolol (10(-6) M) but not by phentolamine (10(-5) M). CoCl2 (1 mM) did not affect the currents. Substitution of choline+ in the recording solution for Na+ abolished the currents, but tetrodotoxin (TTX, 10(-6) M) had no effect on them. The adrenaline-induced currents exhibited a characteristic voltage-dependency: the conductance became large at hyperpolarized membrane potential (-150 to -30 mV) and approached zero at the depolarized membrane potential (greater than -30 mV), but was never reversed up to 30 mV, suggesting that the currents are different from non-specific cation currents. Substitution of isethionate- for Cl- in the recording solution had no effect on the voltage-dependency of the adrenaline-induced currents, whereas substitution of choline+ for Na+ apparently attenuated the voltage-dependency of the currents. These results indicate that adrenaline induces Na(+)-dependent inward currents through activation of beta-adrenergic receptors in bullfrog motoneurons.

Animals

Characterization of antagonistic activity and binding properties of SR 95531, a pyridazinyl-GABA derivative, in rat brain and cultured cerebellar neuronal cells.

Experiments were performed to characterize the antagonistic activity and binding properties of SR 95531 [2-(3' carbethoxy-2'-propyl)-3-amino-6-paramethoxy-phenyl-piridazinium bromide] in rat brain. SR 95531 and bicuculline methiodide inhibited muscimol-stimulated 36Cl- uptake in cortical synaptoneurosomes in a concentration-dependent manner. The inhibitory potency of SR 95531 for the muscimol-stimulated 36Cl- uptake was 15 times higher than that of bicuculline methiodide. Scatchard plots of binding isotherms exhibited two apparent binding sites for [3H]SR 95531 in both the frontal cortex and cerebellum. The IC50 value of SR 95531 for muscimol-stimulated 36Cl- uptake into cortical synaptoneurosomes was in close agreement with the KD value of low-affinity binding sites of [3H]SR 95531 in the frontal cortex. Pretreatment of the membranes with phospholipase A2 invariably decreased [3H]SR 95531 binding in the frontal cortex and cerebellum. On the other hand, the treatment significantly increased [3H]gamma-aminobutyric acid (GABA) binding in a concentration-dependent manner in the frontal cortex. Although lower concentrations of phospholipase A2 did not affect [3H]GABA binding in the cerebellum, treatment with higher concentrations of phospholipase A2 increased the binding in this region. Specific binding of [3H]SR 95531 was also detected in cultures rich in cerebellar granule cells. Pretreatment with phospholipase A2 affected the binding of [3H]GABA and [3H]SR 95531 in these cells, as in the case of the cerebellum. These effects of phospholipase A2 on the binding of [3H]GABA and [3H]SR 95531 were partially prevented by the addition of delipidated bovine serum albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Growth and death behaviour of anchorage-independent animal cells immobilized within porous support matrices.

Growth and death of anchorage-independent animal cells entrapped within porous biomass support particles (BSPs) in static or shake-flask cultures were evaluated by comparison of enzyme activity with non-immobilized cells grown under static culture using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and release of lactate dehydrogenase into the culture medium. Mouse myeloma MPC-11 (ATCC CCL 167) cells inoculated within porous polyvinyl formal resin BSPs (3 x 3 x 3 or 2 x 2 x 2 mm; mean pore diameter, 60 microns) grew exponentially at a specific growth rate comparable to that of non-immobilized cells in the initial period of incubation. Entrapped cells then reached the stationary phase with a cell density over 10(7) cells/cm3 BSP. The death rate of entrapped cells increased in response to the rise in viable cell density in the BSPs. Observation of viable cell distribution within the BSPs using MTT staining indicated that the cells concentrated within a thin outer shell of the BSPs with time. After the immobilized cells reached the stationary phase, penetration of cells into the outer shell ceased and heterogeneous distribution of cell density occurred in the viable cell layer in the shake-flask culture.

Animals

A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes.

To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2 x 2 x 2 mm, mean pore size: 100, 250 or 500 microns) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6-11.3 x 10(7) hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2 x 10(7) cells/cm3 PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes.

Alanine Transaminase

D1 dopamine receptor binding in mood disorders measured by positron emission tomography.

D1 dopamine receptor binding in mood disorders was studied by positron emission tomography (PET) using 11C-SCH23390. Ten patients with bipolar mood disorders and 21 normal controls were studied in the drug-free state. The patients were in euthymic (N = 6), depressed (N = 3) and manic (N = 1) states. Regional radioactivity in the brain was followed for 40 min by PET. A two-compartment model was used to obtain the binding potential (k3/k4) for the striatum and frontal cortex. The binding potentials for the frontal cortex for the patients were significantly lower than those for normal controls, whereas those for striatum were not significantly different. These findings suggest that D1 dopamine receptors in the frontal cortex may be in a different state in patients with bipolar mood disorders.

Adult

Protein-losing enteropathy and pancreatic involvement in a case of connective tissue disease.

A patient with connective tissue disease presenting with both protein-losing enteropathy and pancreatic involvement is reported. A 52-year-old female was admitted because of mild epigastralgia, anasarca and ascites. Serum albumin, transferrin and zinc, showed low levels. An Upper G.I. series and endoscopy showed thickened folds of the duodenum and the jejunum. Biopsy specimens revealed lymphangiectasia in edematous villi. 99mTc-labeled human serum albumin scintigram showed abnormal radioactivity in the small intestine 90 minutes after intravenous injection, indicating protein-losing enteropathy. Hypoalbuminemia was ameliorated by glucocorticoid therapy, but recurred twice when glucocorticoid treatment was tapered. Hypoalbuminemia has not occurred since intestinal lymphangiectasia was improved with glucocorticoid treatment. Levels of elastase 1 and lipase were high in serum and ascites on admission. Endoscopic retrograde pancreatogram showed no abnormalities. Serum pancreatic enzymes were also ameliorated by glucocorticoid therapy, but slightly high levels continued for about one year and a half. This case might have been diagnosed as systemic lupus erythematosus although mixed connective tissue disease was also suspected. There are few reports of protein-losing enteropathy and pancreatic involvement associated with connective tissue diseases. Protein-losing enteropathy and pancreatic involvement were ameliorated with glucocorticoid treatment, suggesting participation of immunological mechanisms.

Connective Tissue Diseases

Sperm immobilizing antibodies react to the 3-O-sulfated galactose residue of seminolipid on human sperm.

It is well known that very few women who possess sperm immobilizing antibodies in their sera can conceive naturally even though there are no abnormalities in their reproductive organs on routine medical examination. A monoclonal antibody (MAb), designated 2H12, was produced by immunizing a BALB/c mouse with the human choriocarcinoma cell line JEG-3. MAb 2H12 showed strong sperm immobilizing activities and reacted to sulfatide and seminolipids. The sperm immobilizing activities of 2H12 were clearly absorbed with sulfatide or seminolipid whilst several other sperm immobilizing MAbs that were made by immunization with human sperm or seminal plasma could not be absorbed with the same sulfoglycolipids. The sperm immobilizing antibodies in the sera of infertile women with unknown cause were also clearly absorbed with sulfatide or seminolipid. MAb 2H12-conjugated immunobeads (MAb 2H12-IMBs) bound to motile sperm. This binding of 2H12-IMBs to sperm was competitively inhibited either by 2H12 or women's sera containing sperm immobilizing antibodies, but not by normal women's sera or several other sperm immobilizing MAbs to human sperm. These results suggest that the sperm immobilizing antibody in women's sera is directed against the 3-O-sulfogalactose residue of seminolipid on the sperm membrane.

Animals