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Biomedical subjects

M Saito

Publications and source records attributed to M Saito.

At least 127 records · Page 7Linked to original sources

Risk factors for development of status epilepticus in dogs with idiopathic epilepsy and effects of status epilepticus on outcome and survival time: 32 cases (1990-1996).

OBJECTIVE: To identify risk factors for episodes of status epilepticus (SE) in dogs with idiopathic epilepsy and determine how SE affects long-term outcome and survival time. DESIGN: Retrospective study. ANIMALS: 32 dogs with idiopathic epilepsy. PROCEDURE: Information on signalment, seizure onset, initiation of treatment, anticonvulsants administered, number of episodes of SE, overall seizure control, and long-term outcome was obtained from medical records and through telephone interviews. Differences between dogs that did and did not have episodes of SE were evaluated statistically. RESULTS: 19 (59%) dogs had 1 or more episodes of SE. Body weight was the only variable significantly different between dogs that did and did not have episodes of SE. Thirteen dogs (9 that did not have episodes of SE and 4 that did) were still alive at the time of the study and were > or = 10 years old. Six of the 19 (32%) dogs that had episodes of SE died of causes directly attributed to the seizure disorder. Mean life spans of dogs that did and did not have episodes of SE were 8.3 and 11.3 years, respectively. Survival time was significantly different between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that a substantial percentage of dogs with idiopathic epilepsy will have episodes of SE. Dogs with greater body weights were more likely to have episodes of SE, and early appropriate seizure treatment did not appear to decrease the risk that dogs would have episodes. Most dogs with idiopathic epilepsy had an expected life span, but survival time was shorter for dogs that had episodes of SE.

Animals↗

Anorectic effect of leptin is mediated by hypothalamic corticotropin-releasing hormone, but not by urocortin, in rats.

Leptin is a key afferent signal that decreases food intake and increases energy expenditure by acting on the specific receptors in the hypothalamus. Corticotropin-releasing hormone (CRH) and its homologous peptide, urocortin, are also known to have a potent anorectic effect when given intracranially. To determine possible involvement of these two peptides in the leptin-induced anorexia, in the present study, food intake was measured in rats pretreated with antibodies against CRH and urocortin. In the control rats without antibody pretreatment, intracerebroventricular (icv) injection of leptin (0.1-1 microg/rat) suppressed nocturnal food intake. The anorectic effect of leptin was substantially attenuated in rats pretreated with icv injection of an anti-CRH antibody, but not with an anti-urocortin antibody. These results suggest that the anorectic effect of leptin is mediated by CRH, but not by urocortin, in the hypothalamus.

Animals↗

Mechanism of urinary tract crystal formation following biphenyl treatment.

Coadministration of biphenyl and KHCO3 in the diet of male rats for 13 weeks produced urine crystals, which, by means of LC-MS/MS analyses, were determined to be composed of the potassium salt of 4-hydroxy-biphenyl-O-sulfate (4-HBPOSK). Biphenyl alone or biphenyl with KCl or NaHCO3 in the diet did not produce urine crystals. It was found that the higher concentration of potassium in the urine and the alkaline pH induced by feeding KHCO3 to rats resulted in the formation of urine crystals of 4-HBPOSK due to 4-HBPOSK solubility being lower in urine than in plasma. Urine crystals of 4-HBPOSK produced hyperplasia of the transitional epithelium of the ureter, ureteral obstruction, and hydronephrosis in the urinary tract.

Animals↗

Theoretical identification of the smallest fullerene, C20.

By using first-principles calculations, we calculate the vibronic fine structure in photoelectron spectra of C-20 clusters. Based on our results, we assign one of the recently observed spectra [H. Prinzbach et al., Nature (London) 407, 60 (2000)] to the fullerene structure and, therefore, confirm the experimental claim that the smallest fullerene is really synthesized.

Journal Article↗

Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta.

The kinesin superfamily motor protein KIF1B has been shown to transport mitochondria. Here, we describe an isoform of KIF1B, KIF1Bbeta, that is distinct from KIF1B in its cargo binding domain. KIF1B knockout mice die at birth from apnea due to nervous system defects. Death of knockout neurons in culture can be rescued by expression of the beta isoform. The KIF1B heterozygotes have a defect in transporting synaptic vesicle precursors and suffer from progressive muscle weakness similar to human neuropathies. Charcot-Marie-Tooth disease type 2A was previously mapped to an interval containing KIF1B. We show that CMT2A patients contain a loss-of-function mutation in the motor domain of the KIF1B gene. This is clear indication that defects in axonal transport due to a mutated motor protein can underlie human peripheral neuropathy.

Amino Acid Sequence↗

Discovery of a non-peptide small molecule that selectively mimics the biological actions of calcitonin.

Calcitonin (CT), a 32-amino acid peptide hormone secreted mainly from the thyroid gland, plays an important role in maintaining bone homeostasis. To discover non-peptide small molecules with biological actions similar to those of CT, a cell-based screening of an in-house chemical library was performed and a pyridone derivative (SUN B8155) was identified. Like CT, it elevated cyclic AMP (cAMP) levels in T47D and UMR106-06 cells which endogenously express human and rat CT receptor, respectively. SUN B8155 also stimulated cAMP formation in cells expressing recombinant human CT receptor, but not in those expressing human parathyroid hormone/parathyroid hormone-related peptide receptor. Accumulation of cAMP in T47D cells was blocked by a selective antagonist of CT receptor, salmon CT(8-32), whereas SUN B8155 did not displace the specific binding of [(125)I]CT to the receptor. Our results suggested that the compound selectively interacts with the CT receptor by a mechanism similar to but probably different from that of CT itself. In rats, intraperitoneal administration of SUN B8155 significantly lowered serum calcium levels, like CT. Our results demonstrate, for the first time, that the biological activities of the newly identified small molecule can mimic that of CT, acting via the CT receptor.

Animals↗

Characterization of three members of murine alpha1,2-fucosyltransferases: change in the expression of the Se gene in the intestine of mice after administration of microbes.

We cloned three members of a GDP-fucose:beta-galactoside alpha1,2-fucosyltransferase (alpha1,2-fucosyltransferase) family, MFUT-I, -II, and -III, from a cDNA of murine small intestine, and determined their enzymatic properties after transfection of the genes into COS-7 cells, and their expression in murine tissues by Northern blotting. MFUT-I, -II, and -III exhibited sequence homology with the human H (78.4%), Se (79.0%), and Sec1 (74.9%) gene products, respectively. COS-7 cells transfected with MFUT-I and -II exhibited alpha1,2-fucosyltransferase activity and the best acceptor substrate for both gene products was GA1 to yield a fucosyl GA1 structure, but no activity was detected in COS-7 cells with MFUT-III. MFUT-II yielded a 3.5-kb mRNA transcript in several tissues, whereas MFUT-I and -III were predominantly expressed in epididymis and testis, respectively. The administration of microbes into germ-free mice resulted in a rapid increase of the MFUT-II gene (Se gene) for the synthesis of fucosyl GA1 in the intestine.

Amino Acid Sequence↗

Up-regulation of uncoupling proteins by beta-adrenergic stimulation in L6 myotubes.

Catecholamine-induced and beta-adrenergic receptor (beta-AR)-mediated thermogenesis in skeletal muscle is a significant component of whole-body energy expenditure. Skeletal muscle expresses uncoupling protein (UCP) 2 and UCP3, which can dissipate the transmitochondrial electrochemical gradient and thereby may be involved in regulation of energy metabolism. We investigated the effects of beta-AR stimulation on UCP2 and UCP3 expression in L6 myotubes. Stimulation of the cells with epinephrine increased the UCP3 mRNA level transiently at 6 h, and also the UCP2 mRNA level at 6-24 h. The stimulatory effects of epinephrine were also observed in the presence of carbacyclin and 9-cis retinoic acid, and mimicked by isoproterenol and salbutamol (beta2-AR agonists), but abolished by propranolol and ICI-118,551 (beta2-AR antagonists). Pharmacological and mRNA analyses revealed the existence of beta2-AR, but not beta1- and beta3-ARs, in L6 myotubes. These results suggested that catecholamines up-regulate UCP2 and UCP3 expression through direct action on the beta2-AR in skeletal muscle.

Adrenergic beta-Agonists↗

Occurrence of gangliosides in the common squid and pacific octopus among protostomia.

Acidic lipids from tissues of the common squid Todarodes pacificus and the pacific octopus Octopus vulgaris were characterized. Hepatopancreatic tissues of both animals had complex compositions of resorcinol-positive acidic lipids, many of which became reactive with cholera toxin B subunit and anti-G(M1) antibody after in situ treatment with sialidase on TLC. One of the major acidic lipids in squid tissue was isolated and examined for its structure. This acidic lipid was identified to be the ganglioside G(D1a) based upon the susceptibility to sialidases of different substrate specificity, characterization of reaction products, and electrospray ionization-mass spectrometry of the lipid. Hepatopancreatic tissues of squid and octopus also contained acidic lipids that reacted with A2B5, a monoclonal antibody specific to c-series gangliosides. Cerebral ganglia of both animals expressed resorcinol-positive acidic lipids, though their compositional patterns differed from the hepatopancreatic tissues. N-Acetylneuraminic acid was identified as the main species in lipid-bound sialic acid in both tissues. The contents of lipid-bound sialic acid in cerebral ganglia were significantly lower than those of hepatopancreatic tissues in both animals. The present study presents the first evidence for the occurrence of gangliosides in protostomia.

Animals↗

Proinsulin C-peptide rapidly stimulates mitogen-activated protein kinases in Swiss 3T3 fibroblasts: requirement of protein kinase C, phosphoinositide 3-kinase and pertussis toxin-sensitive G-protein.

It has been demonstrated that proinsulin C-peptide possesses several biological activities and that its specific binding sites are present on the surface of cell membranes. However, the molecular and cellular mechanisms of C-peptide actions are poorly known. In the present study we examined the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in C-peptide effects. C-peptide induced the phosphorylation of MAPK [p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2] in Swiss 3T3 and 3T3-F442A fibroblasts but not in 3T3-L1 fibroblasts and some other cell lines such as L(6)E(9) muscle cells. In Swiss 3T3 cells, C-peptide-induced phosphorylation of MAPK was dependent on time and concentration, being maximal at 1 min and at 1 nM C-peptide and was accompanied by an increase in MAPK activity and MAPK kinase (MEK) phosphorylation. The MAPK phosphorylation by C-peptide was abolished by treatment with pertussis toxin (PTX) and also with a MEK inhibitor, PD 98059. In addition, MAPK phosphorylation was attenuated by treatment with a phosphoinositide 3-kinase (PI-3K) inhibitor, wortmannin, and with a protein kinase C (PKC) inhibitor, GF109203X, and by down-regulation of PKC by prolonged treatment with PMA. Similar effects of the inhibitors and PTX were found on the MAPK phosphorylation induced by neuropeptide Y. These results suggest that C-peptide activates MAPK through a putative G(i)/G(o)-linked receptor for C-peptide and through PI-3K-dependent and PKC-dependent pathways.

3T3 Cells↗

Isoform-specific regulation of vascular endothelial growth factor (VEGF) family mRNA expression in cultured mouse brown adipocytes.

We have shown that brown adipose tissue (BAT), a thermogenic organ in mammals, expresses high levels of vascular endothelial growth factor (VEGF) mRNA in response to exposure to cold, which may contribute to angiogenesis associated with cold-induced hyperplasia of this tissue. In the present study, we examined mRNA expression of not only VEGF, but also VEGF-B and VEGF-C, recently cloned VEGF isoforms, in vitro using immortal brown adipocytes (HB2) isolated from mouse BAT. HB2 preadipocytes expressed detectable levels of VEGF, VEGF-B and VEGF-C mRNA, but a low level of VEGF. After HB2 cells differentiated into adipocytes, the VEGF mRNA level increased without a noticeable change in the VEGF-B and VEGF-C mRNA levels. When HB2 cells were stimulated by norepinephrine, the VEGF mRNA level increased without a change in that of VEGF-B, while the VEGF-C mRNA level decreased. A marked reduction of VEGF-C mRNA expression was also found when HB2 cells were treated with agonists of peroxisome proliferator-activated receptor gamma (PPARgamma, troglitazone), retinoic acid receptor (RAR, all-trans retinoic acid) and retinoid X receptor (RXR, 9-cis retinoic acid). These results suggest a specific adrenergic mechanism for up-regulation of VEGF expression different from those for other VEGF isoforms, and thereby the major contribution of VEGF to the cold-induced angiogenesis in BAT. In addition, the agonists of PPARgamma, RAR and RXR are suggested to be inhibitory to angiogenesis through the reduction of VEGF-C production.

Adipocytes↗

Enhanced expression of a-series gangliosides in fibroblasts of patients with peroxisome biogenesis disorders.

Peroxisome biogenesis disorders (PBD) are classified into Zellweger syndrome (ZS), infantile Refsum disease (IRD) and neonatal adrenoleukodystrophy. Disturbances in the differentiation of neural cells such as migration arrest are characteristic of PBD. So far the pathogenesis of these disturbances is not clearly understood. We describe an altered metabolism of glycosphingolipids in PBD which has not yet been investigated. We observed an increased amount of a-series gangliosides, GM2, GM1 and GD1a, in the fibroblasts of patients with ZS and IRD. Gangliosides GM1 and GD1a were not present in detectable amounts in normal subjects. A key step in the synthesis of a-series gangliosides is a transfer of GalNAc to ganglioside GM3, so we determined the level of ganglioside GM3 by immunohistochemical methods. We found a granular structure, which was positive toward anti-ganglioside GM3 antibody in the cytoplasm of the patients' fibroblasts. In control cells, the cell membrane was slightly positive toward anti-GM3 antibody. These results may help to clarify the pathogenesis of PBD with respect to the functional roles of glycosphingolipids in cell differentiation, proliferation and apoptosis.

Fibroblasts↗

Halenaquinone, a novel phosphatidylinositol 3-kinase inhibitor from a marine sponge, induces apoptosis in PC12 cells.

In nerve growth factor-treated PC12 cells, 12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-3,6,8,11(2H,12bH)-tetrone (halenaquinone) caused cytotoxicity in a concentration-dependent manner (EC(50) value; 10 microM). Gel electrophoretic DNA analysis of PC12 cells treated with halenaquinone (10 microM) and 11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-[1S-(1 alpha,6b alpha,9a beta,11 alpha,11b beta)]-3H-furo[4,3,2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione (wortmannin) (3 microM) showed a typical apoptotic DNA ladder. In the flow cytometric analysis, halenaquinone caused apoptosis in a concentration- and time-dependent manner (EC(50) value; 10 microM), whereas 2,3-dihydro-12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-6,8,11(12bH)-trione (xestoquinone) with the methylene group at the C-3 position failed to cause apoptosis, suggesting that the carbonyl group at the C-3 position in halenaquinone is important for exerting apoptotic effects in PC12 cells. Phosphatidylinositol 3-kinase was inhibited by halenaquinone (IC(50) value; 3 microM) as well as wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. Halenaquinone inhibited phosphatidylinositol 3-kinase activity at lower concentrations than those at which it induced apoptosis in PC12 cells. These results suggest that halenaquinone causes the death of PC12 cells through an apoptotic process and that the mechanism of halenaquinone-induced apoptosis may be partially explained by the inhibition of phosphatidylinositol 3-kinase activity.

Acetylcysteine↗

Expressed genes in the extraradical hyphae of an arbuscular mycorrhizal fungus, Glomus intraradices, in the symbiotic phase.

To collect extraradical hyphae of arbuscular mycorrhizal (AM) fungi for RNA isolation, a PVDF membrane was laid on the hyphal compartment of a two-compartment culture system of transformed carrot hairy roots and Glomus intraradices. Extraradical hyphae free from host tissue were easily collected, and their RNA was rapidly extracted with a modified acid guanidinium thiocyanate-phenol-chloroform method. A 3'-RACE (rapid amplification of cDNA ends) of a known gene indicated that this protocol enabled the isolation of mRNA molecules as small as 2.3 kb. The cDNA libraries of an AM fungus from the aseptic extraradical hyphae in a symbiotic state were constructed for the first time. Three-fourth of 150 ESTs (expressed sequence tags) indicated low or no similarities to known sequences from other organisms.

DNA, Complementary↗

Gangliosides in rat kidney: composition, distribution, and developmental changes.

Gangliosides in rat kidney were analyzed for their composition, regional distribution, and developmental changes. Renal tissue from 7-week-old rats showed a GM3-dominant pattern with GD3 and several minor ganglioside components including GM4, GM2, GD1a, and an unknown ganglioside (ganglioside X). The tissue also contained c-series gangliosides that included GT3 as the main component with GT2 in a lesser amount. Ganglioside analysis of cortical and medullary regions of renal tissue suggested the restricted localization of some gangliosides. While GM4 and GD3 were enriched in the cortical region, GM2 was distributed mainly in the medullary area. Renal gangliosides showed unique developmental profiles during a period from Embryonic Day 20 (E20) to 7 weeks postnatal. The content of renal gangliosides increased from E20, reached the highest around Postnatal Day 1, and thereafter, decreased rapidly to the adult level. The ratio of N-glycolylneuraminic acid to total sialic acids in gangliosides tended to change in inverse proportion to the amount of total sialic acids. The composition of major gangliosides in renal tissues shifted from GD3-dominant to GM3-dominant patterns with advancing ages. While GM1 was expressed only at early stages of the development, GM4, GM2, and ganglioside X appeared after Postnatal Day 3. The expression of c-series gangliosides was less affected through the period examined. These results suggest that gangliosides may be implicated with development and function of rat kidney.

Animals↗

Protective immunity to Streptococcus mutans induced by nasal vaccination with surface protein antigen and mutant cholera toxin adjuvant.

In this study, mice were immunized nasally with surface protein antigen of Streptococcus mutans serotype c (PAc) and a nontoxic A subunit mutant of cholera toxin (mCT) E112K, as a mucosal adjuvant. Immunization with PAc and mCT elicited significant PAc-specific secretory IgA in saliva and in nasal secretions. Antibody-forming cell (AFC) analysis confirmed the antibody (Ab) titers by revealing significant numbers of PAc-specific IgA AFCs in the submandibular gland and nasal passages. Furthermore, CD4(+) T cells from cervical lymph nodes exhibited significant proliferative responses when restimulated with PAc in vitro. Importantly, mice that were nasally immunized with PAc plus mCT E112K exhibited significantly reduced oral colonization by S. mutans. These results show that nasal administration of PAc and mCT E112K is potentially an effective mucosal vaccine against dental caries and reduces the colonization of S. mutans in the oral cavity.

Adjuvants, Immunologic↗