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Biomedical subjects

Y Nakada

Publications and source records attributed to Y Nakada.

At least 19 recordsLinked to original sources

Forebrain muscarinic control of micturition reflex in rats.

Functional contribution of the cholinergic pathway between the frontal cortex and basal nucleus of Meynert to micturition reflex was investigated. Male Wistar rats were subjected to bilateral lesion of the basal forebrain by ibotenic acid (IA) injection (7.5 microg/rat on each side) (BF rats). Phosphate buffered saline (PBS) was injected into control rats (sham operated rats; SO rats). Cystometrograms were obtained from conscious BF and SO rats 7-10 days after IA/PBS injection. Bladder capacity (BC) of BF rats was significantly smaller than that of SO rats (approximately 43.7%) and was accompanied by decrease in choline-acetyltransferase activity in the frontal cortices. Oxotremorine M, a muscarinic receptor agonist, increased BC in BF rats, while pirenzepine, an M1 muscarinic receptor antagonist, counteracted the effect of the oxotremorine M-induced increase in BC. Injection of oxotremorine M into the dorsal pontine tegmentum (DPT) reduced BC in BF and SO rats, while injection of pirenzepine had no effect on cystometrograms. These findings indicate that the M1 muscarinic receptor plays a part in the forebrain inhibitory mechanisms involved in the micturition reflex and that muscarinic receptor in the DPT contributes to excitatory control of micturition reflex.

Animals↗

Molecular characterization and regulation of the aguBA operon, responsible for agmatine utilization in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) and N-carbamoylputrescine amidohydrolase (encoded by aguB). The aguBA and adjacent aguR genes were cloned and characterized. The predicted AguB protein (M(r) 32,759; 292 amino acids) displayed sequence similarity (< or =60% identity) to enzymes of the beta-alanine synthase/nitrilase family. While the deduced AguA protein (M(r) 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis. The aguR gene encoded a putative regulatory protein (M(r) 24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of the aguBA operon. The presence of an inducible promoter for the aguBA operon in the aguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping. Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background. Interaction of AguR overproduced in Escherichia coli with the aguBA regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine and N-carbamoylputrescine to AguR would antagonize its repressor function.

Agmatine↗

Rapid detection of Brevibacillus formosus BN53-1 in chicken feces.

Here we describe a rapid method for detecting the hydrogen sulfide-decomposing bacterium Brevibacillus formosus BN53-1 in chicken feces. The method, which can be adapted to the specific detection of a variety of useful eubacteria, is based on blot hybridization and polymerase chain reaction (PCR), and makes use of the genus or species hypervariable region of eubacterial 16S rDNA. The approximate limit of detection under the conditions we tested was 1.0 x 10(3) cells in 10 mg of chicken feces.

Animals↗

Antisense oligonucleotides specific to mutated K-ras genes inhibit invasiveness of human pancreatic cancer cell lines.

BACKGROUND/AIMS: Point mutations of the K-ras gene are detected in > 90% of human pancreatic cancers and may play an important role in tumorigenesis. However, correlations between mutant K-ras and the invasive activity of the tumor have remained unclarified. METHODS: 17-merphosphorothioate antisense oligonucleotides targeting K-ras point mutations were transfected into three kinds of human pancreatic cancer cell lines (MIAPaCa-2, PANC-1, and BxPC-3), and the invasive activity was investigated using an in vitro chemoinvasion assay. RESULTS: Antisense oligonucleotides strongly inhibited the invasive activity of the cell lines with mutant K-ras genes (MIAPaCa-2, PANC-1), but not in that with a wild-type K-ras (BxPC-3). CONCLUSION: Antisense oligonucleotides specific to mutated K-ras genes inhibited the invasiveness of human pancreatic cancer cell lines. Specific antisense therapy to the point mutation of K-ras might be a new anticancer strategy for pancreatic cancer.

Blotting, Western↗

Enzyme immunoassay for 6-amino-5-chloro-1-isopropyl-2-(4-methyl-1-piperazinyl)benzimidazole, a novel 5-HT3 receptor antagonist.

An enzyme immunoassay (EIA) has been developed for determination of 6-amino-5-chloro-1-isopropyl-2-(4-methyl-1-piperazinyl) benzimidazole (KB-6806), a novel 5-HT3 receptor antagonist. Anti-KB-6806 antiserum was elicited against the KB-6806-bovine serum albumin (BSA) conjugate prepared by a diazo coupling reaction through the inherent 6-amino group. Beta-galactosidase-labeled 6-amino-5-chloro-1-isobutyl-2-(4-methyl-1-piperazinyl)benzimidazole was similarly prepared by diazo coupling reaction as an enzyme-labeled antigen with a hapten heterologous combination of antiserum. The modification at the 4-methyl group of the piperazine moiety of KB-6806 significantly decreased the binding affinity to the antibody. This method could quantitate KB-6806 in dog plasma in the concentration range of 0.078-10 ng/ml with good accuracy and precision. The EIA method has been successfully applied to the determination of KB-6806 in plasma after intravenous administration of KB-6806 to dogs.

Animals↗

Characterization and chromosomal mapping of a human Necdin pseudogene.

The necdin gene is expressed predominantly in postmitotic neurons and encodes a growth suppressor that interacts with the transcription factors E2F1 and p53. Human necdin gene (NDN) is maternally imprinted and located in Prader-Willi syndrome deletion region 15q11.2-q12. We isolated an NDN homologous sequence from a human genomic DNA library. The homologous sequence is overall 83% identical with necdin cDNA sequence, and possesses a short poly(A) stretch at the 3' end and direct repeats at both ends. Expression of the homologous sequence, which lacks a 5' promoter sequence, was undetected in cultured human cell lines. We mapped this sequence to chromosome 12q14-q21.1 by fluorescence in situ hybridization. These characteristics of the NDN-homologous sequence are consistent with those of processed pseudogenes. The information about the necdin pseudogene in the human genome will be useful for genetic studies on NDN-associated neurogenic disorders.

Base Sequence↗

Ameliorative effects of tamolarizine on place learning impairment induced by transient forebrain ischemia in rats.

In the present study we investigated the effect of (+/-)-1-(3, 4-dimethoxyphenyl)-2-(4-diphenylmethylpiperazinyl) ethanol dihydrochloride (tamolarizine), a calcium entry blocker, on place learning impairment in rats with damage selective to the hippocampal CA1 subfield induced by transient forebrain ischemia. Tamolarizine was administered (40 mg/kg) immediately after 15-min brain ischemia. Place learning was tested in a task in which the rat was required to alternatively visit two places located diametrically opposite each other in an open field. The ischemia+saline group showed severe learning impairment in this task; their performance level was significantly inferior to that of the sham-operated group through the test period (30 days). Although the ischemia+tamolarizine group showed slight impairment of place learning during the course of this test, they later reached almost the same performance level as the sham-operated group. Selective neuronal loss in the CA1 subfield was much less in the ischemia+tamolarizine group than in the ischemia+saline group. These results indicate that tamolarizine treatment protects the hippocampus from ischemic brain damage and ameliorates place learning impairment.

Animals↗

[A case of Lemmel's syndrome with serious jaundice exacerbated by eating].

An 86-year old woman rapidly developed serious jaundice (T-Bil 18.3 mg/dl 12.3 mg/dl). Her jaundice was exacerbated by eating, and improved by fasting. Abdominal CT showed a giant diverticulum in the second part of the duodenum and dilation of the proximal common bile duct. Endoscopic findings confirmed juxtapapillary duodenal diverticulum in contact with the distal common bile duct. MRCP revealed extrinsic compression of the distal common bile duct by the diverticulum. Lemmel's syndrome was diagnosed. Jaundice did not recur after surgery. We speculated that in this case the diverticulum filled by duodenal contents easily compressed the distal common bile duct after eating.

Aged↗

Effects of aniracetam on bladder overactivity in rats with cerebral infarction.

Aniracetam has been used to improve the mental condition of patients with cerebrovascular disease. Previous studies have demonstrated that aniracetam activates the residual functions of cholinergic neurons in damaged brain areas. In this study, the effects of aniracetam on bladder overactivity after left middle cerebral artery occlusion were assessed through oral or i.c.v. administration in sham-operated and cerebral infarcted rats. Oral administration of aniracetam (100 and 300 mg/kg) resulted in a significant and dose-dependent increase in bladder capacity in cerebral infarcted rats but had no effect on bladder capacity in sham-operated rats. Intracerebroventricular administration of aniracetam (0.25 and 2.5 microg/rat) resulted in a significant and dose-dependent increase in bladder capacity in cerebral infarcted rats but not in sham-operated rats. Aniracetam had no significant effect on bladder contraction pressure or micturition threshold pressure in either sham-operated or cerebral infarcted rats. Furthermore, i.c.v. administration of atropine (1 microg/rat), a muscarinic acetylcholine receptor antagonist, completely inhibited the enhancing effects of aniracetam on bladder capacity in cerebral infarcted rats. The effects of aniracetam on bladder overactivity are thought to be mediated in part by activation of cholinergic inhibitory mechanisms in the brain. These results indicate that aniracetam may improve the neurogenic voiding dysfunction observed in patients with cerebrovascular disease.

Animals↗

Subcutaneously inoculated cells and implanted pancreatic cancer tissue show different patterns of metastases in Syrian golden hamsters.

CONTEXT: We studied behavior of the subcutaneously implanted pancreatic tumors and the process of metastasis using syngeneic Syrian golden hamsters. DESIGN: HaP-T1, a cell line derived from nitrosamine-induced pancreatic cancer in Syrian golden hamsters was used for this experiment. Thirty-five animals were divided into two groups: subcutaneous cell inoculation and subcutaneous tissue implantation. The tumor tissue was obtained from subcutaneously implanted cancer cells. One month after implantation, the tumors were resected and studied histopathologically. The animals were followed-up weekly by palpation of the peripheral lymph nodes in order to identify local recurrence. After death, necropsy was performed. Liver, lungs and pancreas specimens were taken for histopathogical study and detection of K-ras point mutation using the PCR/RFLP method. RESULTS: The mean survival time in the subcutaneous cell inoculation group was 151+/-17.5 days, and in the subcutaneous tissue implantation group was 137 +/-12.9 days. During the follow-up, 13 subcutaneously cell inoculated hamsters (86.7%) had right axillary lymph node metastasis while subcutaneously tissue implanted hamsters did not show any palpable lymph nodes. After necropsy, 10 of the 20 subcutaneously tissue implanted animals (50%) showed metastases in the lungs at the histopathological level. However, 16 of the 20 subcutaneously tissue implanted animals (80%) showed K-ras point mutation in the lung specimens. The lungs of the animals of the subcutaneous cell inoculation group did not show any metastases. No metastases were found in the liver or the pancreas in either group. CONCLUSION: This study suggests that homologous subcutaneous cell inoculation and subcutaneous tissue implantation models showed completely different patterns of metastasis. These models may aid further research to clarify the mechanisms of metastasis in pancreatic cancer.

Animals↗

Preparation of nanoparticles consisted of poly(L-lactide)-poly(ethylene glycol)-poly(L-lactide) and their evaluation in vitro.

This study describes the preparation and the evaluation of biodegradable poly(L-lactide)-poly(ethylene glycol)-poly(L-lactide) copolymer (PLA-PEG-PLA) nanoparticles containing progesterone as a model drug. PLA and PLA-PEG-PLA copolymers, whose PEG content ranged from 5.2 to 25.8% (w/w), were polymerized in our laboratory. PEG with weight-average molecular weight (Mw) 6600 or 20 000 was introduced as a hydrophilic segment into a hydrophobic PLA homopolymer. A solvent evaporation method was used to prepare the nanoparticles. The drug trapping efficiencies were around 70% and the weight-averaged mean diameters of the nanoparticles were less than 335 nm. The amount of drug released increased as the PEG content and Mw of PLA-PEG-PLA copolymers increased and the total Mw of copolymers of nanoparticles decreased. The initial burst of drug release was reduced by removing the low Mw fraction from the polymer. During the release test, both the extent to which the copolymers were degraded and the size of the nanoparticles were increased slightly by increasing the content of PEG in the polymers. Drug release from the nanoparticles could potentially be controlled by changing the PEG content, PEG Mw and total Mw of the copolymer. The molecular weight distribution (Mw/Mn, Mn: number-average molecular weight) of copolymers was also an important factor for controlled release.

Biocompatible Materials↗

Purification and properties of hydrogen sulfide oxidase from Bacillus sp. BN53-1.

A hydrogen sulfide oxidase was purified to homogeneity from the heterotroph Bacillus sp. BN53-1 isolated from pig feces compost. The enzyme was found to be a monomer with a M(r) value of approximately 37 kDa. It required FAD for its activity, which was not replaced by FMN. The optimum reaction pH and temperature were 7.5 and 40 degrees C, respectively. The enzyme was stable between pH 6.0 and 7.0 and up to 30 degrees C. Its activity was stimulated by Ca2+ and Mn2+ and inhibited by Al3+, dithiothreitol, and 2-mercaptoethanol. The main product was elemental sulfur, and H2O2 was not detected. The N-terminal sequence of the enzyme showed similarity to other FAD-requiring enzymes.

Journal Article↗

[Epidemiology of subacute sclerosing panencephalitis in Okinawa, Japan, 1970-1996].

In Okinawa, there were ten cases (7 male and 3 female) of subacute sclerosing panencephalitis (SSPE) from 1970 to 1996. All cases had a history of measles and none had received measles vaccination. The incidence of SSPE in Okinawa was 0.31 per million in 1970-1996. Being higher than other districts in Japan. Two cases contracted measles in 1973 and other three in 1990, showing a tendency to cluster.

Age Factors↗

The human chromosomal gene for necdin, a neuronal growth suppressor, in the Prader-Willi syndrome deletion region.

Necdin is a growth suppressor expressed in virtually all postmitotic neurons in the brain. The human necdin gene, NDN, is maternally imprinted and deleted in the Prader-Willi syndrome, a neurobehavioral contiguous gene disorder. Here, we isolated and characterized the human chromosomal necdin gene and its promoter region. The necdin gene is intronless, and it encodes a protein of 321 amino acid residues, four residues shorter than mouse Necdin. By fluorescence in-situ hybridization analysis, the necdin gene was localized to chromosome 15q11.2-q12 within the Prader-Willi syndrome deletion region. CpG islands were found in a region extending from the proximal 5'-flanking sequence to the protein coding region. The 5'-flanking sequence, which lacks canonical TATA and CAAT boxes, possessed a promoter activity in postmitotic neurons derived from murine embryonal carcinoma P19 cells. Methylation in vitro of HhaI CpG sites in the promoter region reduced the transcriptional activity. These results suggest that the necdin gene is silenced through methylation of the CpG island encompassing its promoter region.

Amino Acid Sequence↗

Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides.

Antisense oligonucleotides with base sequences complementary to a specific RNA can, after binding to intracellular mRNA, selectively modulate the expression of a gene. However, these molecules are poorly stable in biological fluids and are characterized by a low intracellular penetration. In view of using oligonucleotides as active molecules, the development of polymeric particulate carriers was considered. Oligonucleotides were associated with biodegradable polyalkylcyanoacrylate nanoparticles through the formation of ion pairs between the negatively charged oligonucleotides and hydrophobic cations. Oligonucleotides bound to these nanoparticles were found to be protected from nuclease attack in cell culture media and their cellular uptake was increased as the result of the capture of nanoparticles by an endocytotic/phagocytotic pathway. The in vivo pharmacokinetic profile of oligonucleotides free or associated with nanoparticles has been investigated after intravenous administration to mice and the stability of these molecules has been evaluated by original methodology based on the use of polyacrylamide gel electrophoresis (PAGE) followed by multichannel radioactivity counting. Stability in vivo in the plasma and in the liver was shown to be improved when the oligonucleotides were adsorbed onto the nanoparticles. These results obtained both in vitro and in vivo open exciting perspectives for the specific delivery of oligonucleotides to the liver, thus considering this approach for the treatment of liver diseases (e.g. liver metastasis or hepatitis).

Animals↗

Unclassified sclerosing bone dysplasia with osteopathia striata, cranial sclerosis, metaphyseal undermodeling, and bone fragility.

Sclerosing bone dysplasias are diagnosed on the basis of a characteristic pattern of osteosclerosis and clinical manifestations; in many of them, cause and pathogenesis are still unknown. A 33-year-old man had five fractures of the humerus, tibiae, and femur as a result of mild traumatic incidents that occurred between the ages of 18 and 33 years as well as a remnant of rib fractures without apparent trauma on radiographs. His height was 158 cm (-2.2 SD). Radiographic evaluation showed cranial sclerosis, longitudinal striations in the metaphyses of the femur and tibia, fan-like striation in the ilium, metaphyseal widening in the femur and tibia, and sclerosis of the ribs. The blood chemistry findings, including serum calcium, phosphorus, and alkaline phosphatase, were normal. Biopsy from the ilium showed thick trabeculae composed of woven bone. The coexistence of osteopathia striata, cranial sclerosis, metaphyseal undermodeling, and bone fragility has not been recognized previously. Our case appears to represent a new form of sclerosing bone dysplasia.

Abnormalities, Multiple↗