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Biomedical subjects

M Nakanishi

Publications and source records attributed to M Nakanishi.

At least 19 recordsLinked to original sources
PubMed1992

Cytosolic and intranuclear calcium signals in rat basophilic leukemia cells as revealed by a confocal fluorescence microscope.

A confocal fluorescence microscope with an argon-ion laser (488 nm) and a He-Cd laser (325 nm) was used to study spatial heterogeneity of the calcium signals in rat basophilic leukemia 2H3 cloned cell line (RBL-2H3). After stimulation with antigen (2,4-dinitrophenol-conjugated bovine serum albumin), fluo-3-fluorescence intensities increased in individual RBL-2H3 cells with different lag times. Time-dependent profiles of the fluo-3-fluorescence intensities resembled closely the patterns of the sequential fluorescence-ratio images of fura-2, which were used to measure the intracellular free-calcium concentration ([Ca2+]i) in individual RBL-2H3 cells using a conventional fluorescence microscope. The present results obtained using the confocal fluorescence microscope showed spatial heterogeneities of fluo-3-fluorescence intensities, suggesting the existence of spatial heterogeneity of [Ca2+]i in RBL-2H3 cells. That is, the results showed that calcium signals first occurred transiently at pseudopodia in RBL-2H3 cells, then the signals transferred to the central parts of the cells. In addition, from the fluorescence images of co-loaded Hoechst 33342 (bisbenzimide H 33342, a DNA-specific probe) which were produced by excitation with a He-Cd laser, it was found that the fluorescence images of the nucleus were quite similar to those of the calcium signals mentioned above. This suggested that the receptor-mediated calcium signals were transferred not only to the cytoplasm but also to the nucleus.

Animals↗
PubMed1992

Single cell observation of ligand-induced desensitization of B-cell membrane immunoglobulin-mediated calcium signals.

Using a digital imaging fluorescence microscope we have observed the membrane immunoglobulin (mIg)-induced desensitization of calcium signals in individual BAL17 B lymphoma cells which express two kinds of antigen receptors, mIgM and mIgD. The mIgD-mediated desensitization was partly abrogated by pretreating the cells with phorbol 12-myristate 13-acetate (PMA) for 24 h, however, the mIgM-mediated one was not affected by the pretreatment. This supports the idea that at least two mechanisms are operative for mIg-induced desensitization in B cells.

B-Lymphocytes↗
PubMed1992

Fluorescence quenching measurements of the membrane bound lipid haptens with different length spacers.

Dansyl lipid haptens with three different length (short, intermediate and long) spacers have been incorporated into DMPC or DPPC liposomes. Anti-DNS-IgG bound most efficiently to these liposomes containing lipid haptens with an intermediate length spacer, although the binding efficiency became more increased when liposomes were made of a mixture of phospholipid (DMPC or DPPC) and cholesterol. To explain these results we have measured the accessibility of dansyl lipid haptens in liposomal membranes by the fluorescence quenching method. It was found that the dansyl haptens located on the surfaces of DMPC (or DPPC) membranes and fluorescence quenchers (iodide ions) possessed almost similar accessibility for the dansyl haptens with different length spacers. However, in the DMPC (or DPPC) membranes with 50% cholesterol, a part of the dansyl haptens became buried into the interior of the liposomal membranes depending on the length of the spacer and another part removed into the aqueous solution with greater affinity for antibody. These results were explained well by our recent model for antibody binding to the membrane-bound lipid haptens.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Changes in the plasma abnormal prothrombin level following treatment of hepatocellular carcinoma.

The Hokkaido Liver Cancer Study Group focused on the changes in PIVKA-II levels observed in 61 HCC patients after several regimens of treatment in comparison with the AFP levels and the pathophysiological characteristics of HCC. The overall positivity rate for PIVKA-II was 47%, and there was no correlation between the PIVKA-II values and the AFP levels. Accordingly, the HCC detection rate was increased by about 20% by the measurement of both markers. In all, 13 patients underwent hepatic resection, and nonsurgical therapy was carried out in the other 48 subjects. Of the 6 surgically treated patients, 5 (83%) showed a fall in PIVKA-II levels to the normal range immediately after surgery, whereas 14/29 (48%) subjects receiving nonsurgical treatment showed a decrease in PIVKA-II values. Although inconsistency between these tumor markers was detected in four treated cases, we concluded that assay for both of these two parameters may expand their clinical utility for the diagnosis of HCC and monitoring of patients after treatment.

Adult↗
PubMed1992

Escherichia coli RecA protein modified with a nuclear location signal binds to chromosomes in living mammalian cells.

We tried to make a well-characterized bacterial protein function in mammalian cell nuclei. For this purpose we chose Escherichia coli RecA protein and fused its carboxy terminus to the nuclear location signal of SV40 large T-antigen by oligonucleotide-dependent modification of the gene. When injected into the cytoplasm, the modified RecA protein (T-RecA for the T-antigen signal) accumulated efficiently in the nuclei, whereas the wild-type RecA protein remained in the cytoplasm. The T-RecA protein retained its original in vivo activity, judging from the finding that uv-sensitive bacteria (recA- E. coli) became uv-resistant on transformation with the T-recA plasmid as well as the recA plasmid. For expression of the T-recA gene in mammalian cells, the 5' region was replaced by the chicken beta-actin promoter and Kozak's initiation signal. A high level of expression was observed when Chinese hamster ovary (CHO-K1) cells were transfected with this plasmid. Indirect immunofluorescence examination revealed that the T-RecA protein in nuclei of mammalian cells bound to chromatin.

Amino Acid Sequence↗
PubMed1992

Growth potential of endometrial cancers assessed by a Ki-67 Ag/DNA dual-color flow-cytometric assay.

Ki-67 is a monoclonal antibody directed against a nuclear antigen present only in proliferating cells. To assess the growth potential of uterine endometrial cancer, the population of cells in proliferating cycle (%PC) was examined with Ki-67, using flow cytometry. The %PC of 27.18 +/- 12.00% in 22 endometrial cancers was significantly higher than the 14.5 +/- 5.94% found in 28 normal endometrial tissues. In premenopausal endometrial tissue, the %PC in the proliferatory phase was significantly higher than the %PC found in the secretory phase. In endometrial cancers, an increase of %PC was found in cases with deep myometrial invasion, and the %PC was elevated in groups containing histologically poorly differentiated types when compared to groups of well-differentiated and moderately differentiated types. Sorted cells reactive with Ki-67 antibody were large and had a high nuclear/cytoplasmic ratio. On the bases of these results, it was concluded that a Ki-67 Ag/DNA dual-color assay would be useful to examine the growth fraction in endometrial carcinoma and that an increased growth fraction was related to deep myometrial invasion or poorly differentiated types.

Adenocarcinoma↗
PubMed1992

A fluorescent molecular rotor probes the kinetic process of degranulation of mast cells.

A confocal fluorescence microscope was used to study the exocytotic secretory processes of mast cells in combination with an fluorescent molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ). DCVJ is known to be an unique fluorescent dye which increases its quantum yield with decreasing intramolecular rotation. Here, DCVJ-loaded peritoneal rat mast cells were stimulated with compound 48/80 and their fluorescence images were compared with fluorescence calcium images of fluo-3-loaded mast cells. Subsequent to transient increases in intracellular free calcium ion concentration, DCVJ fluorescence increased dramatically in the cytoplasm and formed a ring-like structure around the nucleus, suggesting the possibility that the dye bound to the proteins composing the cytoskeletal architecture. Furthermore, the increases of DCVJ fluorescence intensities were mostly blocked in the presence of cytochalasin D (10 microM). However, fluo-3 fluorescence intensities still increased after addition of compound 48/80.

Aniline Compounds↗
PubMed1992

Recognition of the self idiotype by T cells: induction of a rapid increase in cytoplasmic free calcium in T cells recognizing a variable L chain determinant.

To investigate the initial stages of recognition of the self idiotype (Id) by T cells, we examined the early increase in cytoplasmic free calcium ([Ca2+]i) occurring in murine CD4+ T cells specific for a model Id, Id315, following their interaction with the Id. The changes in [Ca2+]i were monitored with stopped-flow fluorometry by loading T cells with fura 2, a Ca(2+)-binding fluorescent dye. An increase of [Ca2+]i in the Id-specific T cell line was dependent on the presence of both antigen-presenting cells (APC) and Id315. When T cells were mixed with APC pulsed with M315 for 90 min at 37 C, a significant increase in T cell [Ca2+]i was observed within one second. A pronounced elevation in [Ca2+]i was also observed in T cells after their interaction with APC which had been pulsed for 90 min with VL-315 Id-containing proteins (such as VL-315, L315, Fv-315 or Fab'-315 fragments). In contrast, pulsing APC for 5 min with the VL fragment produced little or no change in the [Ca2+]i. These results suggest that VL must be further processed by APC before it can be recognized by T cells. Indeed, a synthetic VL region peptide (positions 91-108, designated as P18) produced an elevation in T cell [Ca2+]i when mixed with APC without pulsing.

Animals↗
PubMed1992

Primary immunodeficiency diseases and Epstein-Barr virus-induced lymphoproliferative disorders.

Increased incidence of malignant disorders is noted in patients with both primary and acquired immunodeficiency diseases. The pathogenetic mechanism(s) for these disorders remain unclear. Defective immunosurveillance of these patients, however, is mainly postulated to be responsible for the increased risk of these malignant disorders. Of the malignant disorders, Epstein-Barr virus (EBV)-induced lymphoproliferative disorders (LPD) have been increasingly reported, possibly due to improved therapeutic management techniques such as bone marrow transplantation, which results in prolonged survival periods for the primary immunodeficiency; the dramatic development of immunosuppressive treatments for transplant recipients; and the growing numbers of acquired immunodeficiency syndrome (AIDS) patients. This review focuses on the primary immunodeficiency diseases and EBV-induced LPD, and discusses pathogenetic mechanism(s) for the increased incidence of these malignant disorders.

Adolescent↗
PubMed1992

Immunohistochemical assessment of the growth fraction in cervical cancers using the monoclonal antibody Ki-67; relationship to the clinical stage, histologic type and DNA content.

To assess the growth potential of cervical cancer, cell populations in proliferating cycle (%PC) were examined by an immunohistochemical technique using the monoclonal antibody Ki-67. The %PC was 31.63 +/- 16.61% in 36 cervical cancers and was significantly higher when compared to the 7.8 +/- 3.81% found in 24 samples of normal ectocervical tissues. In cervical cancer tissues, the %PC increased in accordance with progression of the clinical stage of the disease, however, the %PC was not different among the various histologic types of invasive cervical cancers. The DNA index also increased in accordance with progression of the clinical stage of cervical cancer, however, there was no correlation between the %PC and the DNA index. These results suggest that the value of %PC obtained using the monoclonal antibody Ki-67 can be used as a parameter for evaluating the growth activity of cervical cancer and for predicting biological heterogeneity in a tumor.

Aneuploidy↗
PubMed1992

Phenytoin-induced IgG2 and IgG4 deficiencies in a patient with epilepsy.

A five-year-old girl with epilepsy and recurrent respiratory infections was investigated for serum IgG subclass concentrations. She was diagnosed as having a combined deficiency of IgG2 and IgG4 with a decreased serum concentration of IgA and IgG3 and was given replacement therapy with i.v. immunoglobulins. Since then, she has been free from respiratory infections. After phenytoin therapy was stopped, IgG subclass deficiency improved. This case describes the further action of phenytoin on the immune system, adding IgG subclass deficiency to the list.

Child, Preschool↗
PubMed1992

Seroepidemiology status of Nepalese against hemolytic streptococcal infection in Katmandu.

Immune status of apparently healthy Nepalese against hemolytic streptococci was studied by measuring antibodies to various streptococcal antigens such as streptolysin-O (SO), streptokinase (SK), deoxyribonuclease-B (DN-B) and streptococcal polysaccharide (SP). The normal range of anti-streptolysin-O (ASO), anti-streptokinase (ASK), anti-deoxyribonuclease-B (ADN-B) and anti-Streptococcus polysaccharide (ASP) was 60-480 (T.U.), 40-1024 (titer), 60-1280 (titer) and less than 2-128 (Unit), respectively. No difference was observed between the two sexes. ASO and ADN-B were measured by the neutralization method. Passive hemagglutination (PHA) was used to measure ASK and ASP. These values were 2-3 fold higher than those obtained on the Japanese subjects.

Adolescent↗
PubMed1992

Detection of Epstein-Barr virus sequences in patients with Kawasaki disease by means of the polymerase chain reaction.

We used a selective DNA amplification technique to detect Epstein-Barr virus (EBV) DNA in peripheral blood mononuclear cells from patients with Kawasaki disease (KD). By means of the polymerase chain reaction EBV sequences were indentified directly in 21 (60%) of 35 KD patients within 2 weeks after the onset of KD. Furthermore, EBV sequences were detected in all of 6 repeatedly tested patients with KD within 3 months after disease onset. In contrast, only 2 (12%) of 17 control DNA samples were polymerase chain reaction positive. These results indicate that an unusual EBV-cell interaction may exist in KD.

Child, Preschool↗
PubMed1992

Genetic heterogeneity in patients with X-linked recessive chronic granulomatous disease.

Genetic heterogeneity in 12 patients from 11 different families with X-linked recessive chronic granulomatous disease was studied by Southern blot analysis using cytochrome b heavy-chain cDNA as a probe. We found the abnormal restriction length fragment patterns of the cytochrome b heavy-chain gene in three families, which were not observed in healthy controls. DNA from one patient showed the abnormal patterns after digestion with several restriction enzymes. The DNA of two other patients showed the abnormality only with TaqI and PstI. Analysis of the same family members indicated that these abnormal patterns cosegregated with the disease. The other nine patients from eight families did not have any abnormalities detectable by Southern blot analysis. Although further experimentation should be done to study the molecular genetic heterogeneity in most X-linked chronic granulomatous disease families (eight of 11), we were able to demonstrate at least three different types of mutations in the cytochrome b heavy-chain gene responsible for the disease.

Adolescent↗
PubMed1992

The distribution of tissue insulin receptors in the mouse by whole-body autoradiography.

The distribution of insulin binding sites in the mouse was investigated by in vivo whole-body autoradiography. Male mice were injected intravenously with 125I-insulin in the absence of and, in the presence of, excess unlabeled insulin. Three, 6, 15, 30 and 60 minutes after injection, the animals were perfused and subjected to autoradiographic procedures. Specific insulin binding was observed in the choroid plexus, liver, gastrointestinal tract, spleen, pancreas, deferent duct, and Harderian gland. In the liver and spleen, the distribution of binding sites was heterogeneous. In the liver, the density of the binding was higher around the branches of the portal vein than around the central vein. In the spleen, the marginal zone exhibited a higher density than the white and red pulp. The kidney cortex, and the thyroid gland showed a high degree of insulin binding, but the binding was nonspecific. The binding of insulin to other tissues and organs, including the skeletal muscle and fat, was weak, and most of the binding was nonspecific.

Animals↗
PubMed1992

Expression of human metallothionein-II fusion protein in Escherichia coli.

In order to obtain antibody against metallothionein-II (MT-II), a capsid protein metallothionein fusion protein was prepared. A gene encoding human MT-II was cloned into a plasmid for expression of MT fusion protein in Escherichia coli. MT cDNA was generated from human astrocytoma U373MG and amplified by polymerase chain reaction. The nucleotide sequence was identical to reported MT-II. The cDNA was inserted into plasmid pGEM EXTM-2 which carries the T7 promoter and T7 phage 10A major head protein. This expressed phage protein-MT fusion protein, has a molecular weight of 37kDa, forms inclusion bodies and constitutes about 20% of the total protein in transformed E. coli.

Amino Acid Sequence↗