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Biomedical subjects

N Nakamura

Publications and source records attributed to N Nakamura.

At least 19 recordsLinked to original sources
PubMed1992

A novel blocker-PCR method for detection of rare mutant alleles in the presence of an excess amount of normal DNA.

A novel polymerase chain reaction method was developed to preferentially amplify a segment of DNA containing a base substitution mutation. This technique uses a pair of dideoxynucleotide-labeled oligonucleotides (18 mers) of normal sequences as blockers located between the two primers. By virtue of a subtle difference in the melting temperature between the blocker-normal DNA and blocker-mutant DNA hybrids, the method allows preferential amplification of the mutant DNA. We used the human N-ras gene as a model. Two different types of N-ras mutations could be effectively amplified when they were present with an excess amount of normal DNA at a ratio of 1:10(3). Furthermore, the sensitivity was increased 10-fold by using single strand conformation polymorphism analysis for the amplified products, and mutant DNA was detected in the presence of a 10(4) times excess amount of normal DNA.

Alleles↗
PubMed1992

Purification and characterization of a vimentin-specific protease in mouse myeloid leukemia cells. Regulation during differentiation and identity with cathepsin G.

Strong vimentin-degrading activity was found in a mouse myelomonocytic leukemic cell line, M1. When M1 cells were induced to differentiate into macrophage-like cells, this degrading activity decreased, while expression of the vimentin gene increased as reported previously [Tsuru, A., Nakamura, N., Takayama, E., Suzuki, Y., Hirayoshi, K. and Nagata, K. (1990) J. Cell Biol. 110, 1655-1664]. This activity was not due to calpain, which was reported to degrade vimentin, because it was independent of the presence or absence of Ca2+. This activity was revealed to be strongly associated with membranes by differential-centrifugation experiments. To identify this protease, purification of the degradation enzyme was performed. A membrane fraction was prepared and extracted with a buffer containing Triton X-100, then subjected to column chromatography using carboxymethyl-Sepharose and heparin-Sepharose. Quantitative analysis using the purified protease revealed that the specificity of this protease was more than 1000-fold higher for vimentin than for bovine serum albumin, ovalbumin and actin. Four protein bands expressing the activity were finally identified by SDS/PAGE. Amino-terminal sequences of these four proteins were identical, suggesting lower-molecular-mass proteins were degradative products. Furthermore, it was revealed that the sequence had the highest similarity with that of human cathepsin G. This result was consistent with the cathpsin-G-like properties of the purified protease, such as the optimum pH and the specificities for inhibitors. The purified protease degraded a synthetic substrate for cathespin G, succinyl-alanyl-alanyl-prolyl-phenylalanyl-p-nitroanilide, with a comparable specific activity to human cathespin G and was specifically detected with anti-(human cathepsin G) serum in immunoblot analysis. The purified protease thus belongs to the 'cathepsin G family', and perhaps is a mouse homologue of human cathepsin G.

Amino Acid Sequence↗
PubMed1992

Unique association of p53 mutations with undifferentiated but not with differentiated carcinomas of the thyroid gland.

Thyroid neoplasms show a wide variety of lesions varying from slowly growing differentiated adenocarcinomas to rapidly proliferating undifferentiated carcinomas. There has been some histopathological evidence that the undifferentiated thyroid carcinomas are derived from differentiated carcinomas. Moreover, it is suspected that some genetic events might be associated with such changes. In the present study, mutations in the p53 gene were investigated by direct sequencing analysis after polymerase chain reaction amplification of exons 5 to 8, using paraffin-embedded primary tumors and cultured cells. No mutations in exons 5 to 8 were detected in 10 differentiated papillary adenocarcinomas, whereas 6 of 7 undifferentiated carcinomas were found to carry base substitution mutations. Sequencing analysis confirmed mutations at codons 135 (TGC----TGT), 141 (CCC----CCT), 178 (CAC----GAC), 213 (CGA----TGA), 248 (CGG----CAG, CGG----TGG), and 273 (CGT----TGT). The spectrum of mutations (G:C to A:T transitions in 7 of 8) might be a specific feature of the spontaneous cancers. The results strongly suggest that, in human thyroid glands, p53 mutations play a crucial role in the progression of differentiated carcinomas to undifferentiated ones.

Adenocarcinoma, Papillary↗
PubMed1992

Identification of a negative regulatory element involved in tissue-specific expression of mouse renin genes.

The 5' flanking region of the mouse renin genes (Ren-1d and Ren-2d) contains two motifs that are homologous to known negative regulatory elements (NREs). Ren-2d has a 150-base-pair (bp) insertion 5' to the upstream putative NRE (NRE-1), which is lacking in Ren-1d. We tested the functionality of these sequences by using site-directed mutagenesis to delete individually each putative NRE from Ren-1d and to delete the 150-bp insertion from Ren-2d. We examined the effect of these mutations on the expression of the reporter gene chloramphenicol acetyltransferase, which was expressed from a truncated thymidine kinase promoter fused to the renin regulatory region. This plasmid was transfected into human choriocarcinoma JEG-3 cells. Only the upstream NRE (positions -619 to -597) was found to be functional in Ren-1d. The deletion of a 150-bp insertion from Ren-2d resulted in the suppression of chloramphenicol acetyltransferase activity to the level of Ren-1d expression. These data suggest that the upstream NRE that is functional in Ren-1d, but not in Ren-2d, may be partly responsible for differential expression of the renin genes in various tissues. The molecular mechanism of the NRE was examined by studying its interaction with nuclear proteins in submandibular gland and JEG-3 cells by gel-mobility-shift assays. Specific nuclear protein binding was observed only to the upstream NRE and the molecular mass of this protein was approximately 72 kDa as determined by Southwestern blot analysis. Thus our results suggest that both Ren-1d and Ren-2d conserve a cis-acting NRE in the 5' flanking region. In Ren-1d, this NRE could bind a specific nuclear protein resulting in the inhibition of Ren-1d expression in these tissues. On the other hand, the NRE in Ren-2d is nonfunctional due to interference by an adjacent 150-bp insertion.

Animals↗
PubMed1992

A simple and rapid method for HLA-DQA1 genotyping by polymerase chain reaction-single strand conformation polymorphism and restriction enzyme cleavage analysis.

A simple and rapid method for identification of alleles at the human leucocyte antigen (HLA)-DQA1 locus is described. The polymorphic second exon of the HLA-DQA1 locus was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was analyzed by single-strand conformation polymorphism (SSCP) and restriction enzyme cleavage assay. Using this method, the eight known DQA1 alleles could be distinguished from each other. This paper suggests that the method can be used for quick genotyping of DQA1 alleles, but detecting point mutations at various positions in a fragment as well as new HLA-DQA1 genotypes should also be possible.

Alleles↗
PubMed1992

Bone mineral density in the proximal femur and hip fracture type in the elderly.

Bone mineral density (BMD) in the contralateral proximal femur in 100 female elderly patients with hip fracture and the 35 controls without hip fracture were investigated using dual-photon absorptiometry. The hip fracture patients were divided into intracapsular fracture (n = 53) and extracapsular fracture (n = 47) groups, and these two groups were further divided into five subgroups according to fracture site: intracapsular fracture type 1 (transcervical fracture, n = 29) and type 2 (subcapital fracture, n = 24); extracapsular fracture type 1 (intertrochanteric line fracture, n = 13), type 2 (pertrochanteric fracture, n = 28), and type 3 (combined type of pertrochanteric and subtrochanteric fracture, n = 6). The intracapsular fracture group showed BMD values similar to those of controls; the extracapsular fracture group showed significantly lower BMD values than controls. When these two were subclassified into five subgroups, different results were seen in terms of BMD value in the proximal femur and fracture types; intracapsular fracture type 1 showed BMD values equivalent to those of controls; on the other hand, type 2 showed significantly lower BMD value than controls, and the BMD distribution in the proximal femur among the extracapsular fracture subgroups 1-3 differed, although all of them showed significantly lower BMD values than controls. The degree of trauma causing the fractures was also assessed according to available anamnestic data, but no significant difference was found in trauma tendency between the intra- and the extracapsular fracture group or among the subgroups in each group.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorptiometry, Photon↗
PubMed1992

Frequency of mutant T lymphocytes defective in the expression of the T-cell antigen receptor gene among radiation-exposed people.

The frequency of mutant T lymphocytes defective in T-cell receptor gene (alpha or beta) expression was measured using the 2-color flow cytometric technique. Results for a total of 203 atomic bomb survivors, 78 of whom were proximally exposed (DS86 doses of greater than or equal to 1.5 Gy) and 125 of whom were distally exposed (DS86 dose of less than 0.005 Gy), showed that the mutant frequency was significantly higher in males than in females. No significant dose effects were observed. In contrast, a significant increase of mutant frequency was observed for 6 patients treated with Thorotrast, a contrast medium containing thorium-232 formerly used for radioligands. In addition, thyroid disease patients treated with 131I showed a dose-related increase of mutant frequency. It was suggested that the present T-cell receptor mutation assay has a unique characteristic as a biological dosimeter for measurement of recent exposures to genotoxic agents.

Adult↗
PubMed1992

Development of a flow-cytometric HLA-A locus mutation assay for human peripheral blood lymphocytes.

A flow-cytometric technique was developed to measure the frequency of variant lymphocytes lacking expression of HLA-A2 or A24 allele products among donors heterozygous for HLA-A2 or A24. It was found that the variant frequency of lymphocytes in peripheral blood was of the order of 10(-4) and increased with donor age. Molecular analyses of mutant clones revealed that about one-third were derived from somatic recombinations and that the remaining two-thirds did not show any alterations after Southern blotting analysis. In contrast, mutants obtained after in vitro X-ray mutagenesis study were found to be mostly derived from large chromosomal deletions. A small-scale study on atomic bomb survivors did not show a significant dose effect.

Adult↗
PubMed1992

The potentiating effect of KC-404 on prostacyclin-induced vasodilation in isolated human cerebral artery.

1. KC-404 (10(-9)-10(-6) g/ml) produced concentration-dependent relaxations in human middle cerebral arteries contracted with prostaglandin F2 alpha. 2. KC-404 (10(-8) and 10(-7) g/ml) has the ability to significantly potentiate prostaglandin I2-induced relaxations in human middle cerebral arteries. 3. KC-404, at least in low concentrations, may elicit human cerebral vasodilation predominantly by potentiating relaxant responses to prostaglandin I2 produced spontaneously in vascular wall. 4. KC-404 is expected to increase cerebral blood flow at least by dilation of major cerebral arteries, and this agent may be beneficial for the treatment of cerebrovascular disorders.

Cerebral Arteries↗
PubMed1992

Four cases of metastatic lymphoma into the temporal bone.

A histopathological study on 17 temporal bones obtained from 9 patients who died of malignant lymphoma revealed metastasis of malignant lymphoma in 7 temporal bones from 4 of them. There were no differences in stages at the initial diagnosis between the cases with metastasis and those without metastasis. However, the higher was the frequency of metastasis, the longer became the period from the first medical examination to death. In addition, malignant lymphoma was considered to metastasize into the temporal bone by the following two routes, 1) direct infiltration or invasion from the cerebrospinal fluid and 2) hematogenous metastasis or invasion.

Adult↗
PubMed1992

Penetrating injuries to the oropharynx.

A review was made of 12 cases of penetrating injuries to the oropharynx seen at Musashino Red Cross Hospital during the period from 1984 to 1988, and their records were compared with those of past cases with similar injuries reported in a survey for the period between 1949 and 1959. Most of the patients were children under the age of four and were injured when they fell with a sharp-edged object in the mouth. Penetrating injuries to the oropharynx occasionally cause severe complications, including retropharyngeal abscess and mediastinitis. This type of injury should be called a 'pencil injury'.

Age Factors↗
PubMed1992

Reduction of delayed-type hypersensitivity by the injection of n-3 polyunsaturated fatty acids in mice.

The effects of injection of n-3 polyunsaturated fatty acids (PUFAs) on the delayed-type hypersensitivity (DTH) response was investigated in mice. Mice were immunized with sheep red blood cells (SRBCs). Six days later 50 microliters of a 20% SRBC suspension was injected into the right hind footpad of each mouse. Just before the challenge of SRBCs, various amount of a trieicosapentaenoyl-glycerol emulsion (10%) was injected through tail veins (5 mice per each dose). Then 24 hr later the dorsoventral thickness of the right hind footpad was measured and compared with that of the left hind footpad. The difference in thickness between both footpads was regarded as the DTH response. The effect of the emulsion on DTH was dose-dependent; the DTH responses (in mm) in the control group (injected with 0.5 ml of a 2.5% glycerol solution through tail veins) and EPA-injected groups (with 5 mg, 10 mg, and 20 mg) were 1.53 +/- 0.16 (mean +/- SEM), 1.09 +/- 0.14, 0.43 +/- 0.07 (P less than 0.005), and 0.36 +/- 0.13 (P less than 0.005), respectively. The DTH response was also depressed by the injection of a tridocosahexaenoyl-glycerol emulsion. Consequently, n-3 PUFA emulsions have clinical implication in DTH-related diseases such as rejection of allografts.

Animals↗
PubMed1992

Effects of malnutrition on development of experimental pressure sores.

Using food-deprived rabbits we investigated the effects of nutritional deficiency on the development of pressure sores. The body weight of these animals was decreased significantly from normal. Organ weights of liver, heart, spleen, kidney, and testis were significantly decreased from normal as well. Protein deficiency in these animals was indicated by serum tests, as well as by histologic features of liver and testis and ultrastructural findings on fibroblasts. We produced lesions in malnourished as well as normal rabbits by exposing their skin to a balloon-produced compressive force of 120 +/- 10 mmHg for 4 hours. Biopsies were taken 1, 2, and 3 days after the pressure application. Histologic findings at each time were as follows: At day one, the degree of ischemic skin destruction in the malnourished animals was more severe than that in the normal ones, and thrombi were occasionally seen in the malnourished cases. At day two, proliferation of fibroblasts and macrophage infiltration were evidenced in the normal animals, whereas signs of collagen fiber degeneration as well as microthrombi were seen in the malnourished ones. At three days, epidermal cells covered the lesions in the case of normal animals; however, massive necrosis of the epidermis was still recognized in the malnourished rabbits. Thus, the healing process of pressure sores was strongly suppressed in the malnourished animals. This suppression was attributed to the reduction in fibroblast proliferation, capillary formation, macrophage infiltration, and also to the low level of epidermal cell proliferation.

Animals↗
PubMed1992

Disinfection of drinking water by using a novel electrochemical reactor employing carbon-cloth electrodes.

A novel electrochemical reactor employing carbon-cloth electrodes was constructed for disinfection of drinking water. Escherichia coli K-12 (10(2) cells per cm3) was sterilized when a cell suspension was passed through the reactor at a dilution rate of 6.0 h-1, and a potential of 0.7 V versus a saturated calomel electrode was applied to an electrode. The survival ratio increased with increasing dilution rate but was less than 0.1% at dilution rates of less than 6.0 h-1. Although the survival ratio increased with increasing cell concentration above 10(3) cells per cm3, the disinfection rate also increased. The disinfection rate was 6.0 x 10(2) cells per cm3 per h at a cell concentration of 10(2) cells per cm3. Continuous sterilization of E. coli cells was carried out for 24 h. Sterilization is based on an electrochemical reaction between the electrode and the cell which is mediated by intracellular coenzyme A. Sterilization of drinking water by using this reactor was successfully performed, demonstrating the potential of such a reactor for clean and efficient water purification.

Disinfection↗
PubMed1992

Cell-dependent posttranslational processing and secretion of recombinant mouse renin-2.

In the DBA/2 mouse submandibular gland (SMG), renin is predominantly the expression product of the renin gene Ren-2d. Prorenin is synthesized and rapidly converted to a constitutively secreted single-chain intermediate, which is then processed to and stored as the mature two-chain (2C) form, which is released by regulated secretion. To evaluate whether the mode of renin processing is cell dependent, renin (Ren-2d) complementary DNA was stably integrated in the genome of Chinese hamster ovary (CHO) cells and a mouse pituitary cell line (AtT-20) by transfection, and renin processing and secretion were examined. Transfected CHO cells secreted exclusively prorenin, whereas transfected AtT-20 cells secreted both prorenin and active renin. AtT-20 cells processed prorenin to the single-chain polypeptide (1C-renin) that was the main storage form and was not further processed to the 2C form of correct size, whose site of generation or function is uncertain at this time. In addition, the conversion of prorenin to 1C-renin was much slower in AtT-20 cells than in the SMG. Thus the patterns of renin biosynthesis and secretion in AtT-20 cells show major differences when compared with these processes in the native SMG, suggesting that cell-dependent characteristics, e.g., the presence of specific processing enzymes, are important factors influencing mouse renin processing.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Tissue specificity of renin promoter activity and regulation in mice.

Certain mouse strains (e.g., DBA/2) contain two renin genes (termed Ren-1 and Ren-2) and express higher renin levels in nonkidney tissues than strains with a single renin gene. The 5'-flanking regions of the Ren-1 and Ren-2 genes contain several TATA boxes preceding putative transcriptional start sites. These initiators are termed P1a, P1, P2 (from 5' to 3'), and their function (with the exception of P2) is largely unknown. In this study, we mapped the renin transcriptional start sites in renal and extrarenal tissues [adrenal, brain, testis, heart, and submandibular gland (SMG)] and examined the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on tissue specific promoter usage. Our results showed that, in the unstimulated state, P2 (the predicted initiator) is active in all DBA/2 mouse tissues. Additional transcriptional start sites were detected in the adrenal and testis (originated by P1a and P2) and the SMG (originated by P1a, P1, and P2). The administration of 8-bromoadenosine 3',5'-cyclic monophosphate led to selective stimulation of P1a in the adrenal but did not affect the selective usage of initiation sites in other organs. A locus-specific ddNTP primer extension assay was used to verify which renin gene is induced by cAMP. Results indicated that both Ren-1 and Ren-2 responded to cAMP treatment in identical fashion. Taken together, these data indicate that more than one form of renin transcript is present in several mouse tissues. There is tissue specificity in promoter usage in the unstimulated state and in response to cAMP.

Animals↗