Biomedical subjects

Mamoru Nakanishi

Publications and source records attributed to Mamoru Nakanishi.

30 records · Page 2Linked to original sources

PubMed2003

Cholesterol depletion inhibits store-operated calcium currents and exocytotic membrane fusion in RBL-2H3 cells.

The effects of cholesterol depletion from the plasma membrane with methyl-beta-cyclodextrin (MbetaCD) on exocytotic processes were investigated in rat basophil leukemia cells (RBL-2H3 cells). Pretreatment of the cells with MbetaCD inhibited antigen-evoked exocytotic release dose-dependently. To elucidate the mechanism of this inhibition, we performed experiments on the effects of MbetaCD on exocytotic membrane fusion and mobilization of Ca(2+) and on the localization of the tyrosine kinase Lyn. Inhibition of degranulation by MbetaCD was observed even under stimulation with the phorbol ester and calcium ionophore. Therefore, MbetaCD affected a process downstream of Ca(2+) influx, or membrane fusion between the granule and the plasma membrane. Intracellular calcium measurements revealed that MbetaCD inhibited the Ca(2+) increase induced by antigen. Furthermore, we found that MbetaCD significantly inhibited Ca(2+) influx from the extracellular medium through the store-operated calcium channel (SOC) but did not affect Ca(2+) release from the intracellular Ca(2+) store. Fluorescent image analysis of cells expressing Lyn-YFP showed that treatment with MbetaCD scarcely affected the localization and lateral mobility of Lyn in the plasma membrane. These results suggest that cholesterol depletion by MbetaCD decreases degranulation mainly by inhibiting the SOC and membrane fusion between the secretory granules and the plasma membrane in mast cells.

Animals↗

PubMed2003

Remarkable induction of apoptosis in cancer cells by a novel cationic liposome complexed with a bcl-2 antisense oligonucleotide.

We reported recently a novel cationic cholesterol derivative with a hydroxyethylamino head group, cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine (I) for liposome-mediated gene transfection [FEBS Lett., 408 (1997) 232]. In the present paper we have studied whether this novel cationic liposome is prominent in nature to suppress cell growth of human cancer cells. Bcl-2 antisense phosphorothioate oligonucleotides (AS-ODNs) were complexed with the cationic liposomes with the derivative (I) and they were introduced into human cervix epithelial carcinoma cell lines HeLa, and mouse fibroblast NIH3T3 cells. An AS-ODNs targeting/bcl-2 gene induced probably apoptosis (including necrosis in some cases) in HeLa and NIH3T3 cells, however, nonsense oligonucleotides (NS-ODNs) corresponding to a scrambled-sequence control hardly induced apoptosis. Induction of apoptosis was much greater than that by commercially available DC-Chol liposomes. Fluorescence intensities of FITC-conjugated bcl-2 AS-ODNs were specifically found in the nucleus. The intensity of the AS-ODNs was mostly consistent with the amounts of Bcl-2 proteins observed by Western blot analysis in the target cells. The results showed the possibility that this new cationic cholesterol derivative might be very promising to be used for liposome-mediated gene targeting in vitro and in vivo.

Apoptosis↗

PubMed2003

Milnacipran plasma levels and antidepressant response in Japanese major depressive patients.

The relationship between antidepressant effects and plasma levels of milnacipran was studied in 49 cases of major depression without psychotic features during 6 weeks of milnacipran treatment. The daily dose of milnacipran was 50 mg/day for the first week, and up to 100 mg/day thereafter. Depressive symptoms were evaluated by the Montgomery and Asberg depression rating scale (MADRS) before treatment and at 1, 2, 4 and 6 weeks after the beginning of this study. Thirty-four patients (69.4%) were responders (defined as a 50% or greater decrease in the baseline MADRS score). Significant differences of MADRS scores were seen from 1 week after the beginning of this study (p=0.004, unpaired t-test) between responders and nonresponders. The mean plasma milnacipran level of responders, 82.0 +/- 29.4 ng/ml, was similar to that of non-responders, 78.6+/-23.1 ng/ml; there was no significant difference between responders and nonresponders. Neither a significant linear nor a curvilinear relationship was obtained between the final MADRS score and the plasma levels of milnacipran. Although there was no significant relationship between the plasma levels of milnacipran and the antidepressant response, milnacipran should be considered an efficacious agent in the treatment of major depressive patients.

Adrenergic Uptake Inhibitors↗

PubMed2003

Asialoganglioside enhances the efficiency of gene transfection mediated by cationic liposomes with a cationic cholesterol derivative.

We investigated the transfection efficiency mediated by asialoganglioside-containing cationic liposomes. Previously we reported that monosialoganglioside GM(1) (GM(1a)) enhanced transfection efficiency. In this study, we investigated the effects of sialic acid in gangliosides on transfection efficiency. Two mammalian culture cell lines HeLa and HepG2 were transfected with luciferase plasmids (pGL3) using cationic liposomes which contain monosialoganglioside GM(1) (GM(1a)) or its asialic counterpart, asialoganglioside GM(1) (GA(1)). Both GM(1a) and GA(1) enhanced the efficiency of transfection mediated by cationic liposomes, and GA(1) exhibited higher efficiency than GM(1a) in both cell lines. Transfection efficiency of ganglioside-containing liposomes was also assessed by the effects of antisense oligonucleotides (AS-ODN) for bcl-2 gene, which suppresses apoptotic cell death. Western blotting analysis revealed that the expression of Bcl-2 was decreased by AS-ODN, and the reduction of protein expression in cells treated with GA(1)-containing liposomes was more remarkable than that with GM(1a)-containing liposomes. Furthermore, the induction rate of apoptosis was higher in cells treated with AS-ODN with GA(1)-containing liposomes. Together with the results obtained by luciferase assay mentioned above, the removal of sialic acid from ganglioside causes the enhancement of efficiency of transfection mediated by cationic liposomes.

Apoptosis↗

PubMed2003

[Reduction in n-3 PUFA levels and EPA/AA ratio in serum after desert travels in China].

OBJECTIVES: The effects of 3 months of desert travel in China on serum fatty acids and tocopherol were studied. METHODS: In project staff members (6 males, 3 females, aged 19-27 years), serum levels of fatty acids and alpha-tocopherol were analyzed before and after travel by gas liquid chromatography and high-performance liquid chromatography, respectively. RESULTS: Comparison of the levels before and after the trip showed no differences in serum total cholesterol, triglycerides, total protein or alpha-tocopherol. There were no changes in the levels of total fatty acids, while the percentage of polyunsaturated fatty acids increased (p < 0.05). Levels of n-3 PUFA lowered from 166 micrograms/ml to 103 micrograms/ml, and those of n-6 PUFA had increased from 988 micrograms/ml to 1140 micrograms/ml after the trip (p < 0.01 and p < 0.001, respectively). No change was observed in the serum levels of alpha-linolenic acid (C18:3n-3), but lowering of the levels of eicosapentaenoic acid (EPA, C20:5n-3) from 41.4 micrograms/ml to 16.3 micrograms/ml and docosahexaenoic acid (DHA, C22:6n-3) from 107.8 micrograms/ml to 71.7 micrograms/ml was found after the trip (p < 0.05 and p < 0.01, respectively). Serum levels of linoleic acid (LA, C18:2n-6) increased from 832 micrograms/ml to 598 micrograms/ml (p < 0.001), and arachidonic acid (AA, C20:4n-6) tended to increase. The ratios of n-3/n-6 PUFA and EPA/AA decreased from 0.171 to 0.091 and from 0.258 to 0.096 after the trip, respectively (p < 0.01 for both). CONCLUSIONS: Our findings indicated that 3 months of desert travel increased the serum levels of n-6 PUFA and LA and reduced the serum levels of n-3 PUFA and EPA and the ratios of n-3/n-6 PUFA and EPA/AA, possibly due to a relative essential fatty acid deficiency.

Adult↗

PubMed2003

New strategy in gene transfection by cationic transfection lipids with a cationic cholesterol.

The present article reviews interesting cationic liposomes (cationic transfection lipids) with novel cationic cholesterol derivatives, a new strategy in gene transfection developed by our group and the presently accepted molecular mechanism of gene transfection. Use of confocal laser scanning microscopy and atomic force microscopy in elucidating the molecular mechanism of gene transfection by cationic liposomes is also reviewed using examples from our own work. As delineated below, both the confocal laser scanning microscopic and the atomic force microscopic results advocate for the involvement of the sequential three steps in gene transfection mediated by the cationic liposomes: endocytotic internalization of the lipoplexes (liposome-DNA complexes) into the target cells, endosome-lysosome fusion whereby the DNA gets released from the liposomes and moves towards the nucleus of the target cells and microtubule organization apparently involved in trafficking the transfected foreign genes to lysosomes. Furthermore, the present article also reviews couple of important strategies in gene transfection namely, use of liposomes made from biosurfactants and harnessing efficient gene transfection by activating the membrane-bound receptor molecules.

Animals↗

PubMed2003

The ratio of serum paraoxonase/arylesterase activity using an improved assay for arylesterase activity to discriminate PON1(R192) from PON1(Q192).

Human serum paraoxonase (PON1) exists in 2 major polymorphic forms: Q (glutamine) or R (arginine) at codon 192. The PON1(192) activity polymorphism is substrate dependent. The PON1(Q192) isoform has a higher rate of in vitro hydrolysis of diazoxon, sarin, and soman, whereas the PON1(R192) isoform has higher activity for the hydrolysis of paraoxon and chlorpyrifos oxon. Both isoforms hydrolyze phenyl acetate at approximately the same rate. The present study described and evaluated a kinetic method of arylesterase activity determination with a modified fixed incubation method that used the oxidative coupling of phenol with 4-aminoantipyrine of phenyl acetate as the substrate. Our improved method shows that arylesterase activity is lower with the PON1(R192) isoform than with the PON1(Q192) isoform. The average activities of serum of individuals of a specific PON1(Q192) genotype showed higher arylesterase and lower paraoxonase activity than the PON1(R192) genotype. The ratio of paraoxonase/arylesterase activity showed a clear separation of all three PON1(192) genotypes with no overlap between the groups (QQ: < 5.0, QR: 5.0-11.0, RR: > 11.0). PCR has suggested that the PON1(192) phenotypes correspond to the PON1(192) genotypes. Therefore, when conducting epidemiological or mechanistic studies that examine the role of PON1 in organophosphorus or lipid metabolism, this ratio is more useful and informative than a PCR-based genotype alone.

Adult↗

PubMed2003

Study on physical and some serum parameters of inhabitants living in agricultural Terai region, Nepal.

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltranspeptidase (gamma-GTP), lactate dehydrogenase (LDH), urea nitrogen (BUN), uric acid (UA) and creatinine (CRE) levels were examined in 183 people (98 males and 85 females aged 10 to 68 years) living in Terai region in Nepal. The mean values of serum components examined did not differ by sex in the age group of 10-14 years. The mean values of serum AST, ALT and gamma-GTP levels differed significantly between the sexes (P<0.01). The all physical measurements and serum parameters observed correlated well in males, but a few of them correlated in females. The AST-ALT, AST-gamma-GTP, ALT-CRE and BUN-CRE correlated well in both sexes. The UA correlated with ALT and gamma-GTP, CRE with gamma-GTP, LDH and UA in males, while only AST-LDH and gamma-GTP-BUN correlated in females. The levels of most of the serum components examined in this study were within normal ranges for Japanese and others. However, it seemed to be necessary to improve their living conditions as these serum components are related to hepatic or renal function and the infectious diseases.

Adolescent↗

PubMed2002

Transbilayer asymmetry of phospholipids in the plasma membrane regulates exocytotic release in mast cells.

To investigate the role of the asymmetric distribution of phospholipids of the plasma membrane in exocytosis, we examined the effects of disruption of this asymmetrical distribution of lipids on exocytotic release from mast cells (RBL-2H3). Lipid scramblase, which is activated by divalent cations and catalyzes the transbilayer movement of phospholipids, was overexpressed in mast cells. Exogenous lipid scramblase was expressed in the plasma membrane and the cytoplasm. Activation of scramblase by divalent cations disrupted the asymmetrical distribution of phospholipids in the plasma membrane. Exocytotic release induced by calcium ionophore and phorbol ester was significantly inhibited in the cells transfected with wild-type scramblase. This inhibition was observed with time lag of about 5 min. Furthermore, when the asymmetric distribution of lipids was disrupted before induction of exocytosis, the inhibition of exocytotic release was obvious from the beginning without time lag. These results suggest that the asymmetric distribution of phospholipids in the plasma membrane plays an essential role in fusion between secretory granules and the plasma membrane. This finding also demonstrates that the transbilayer asymmetry of phospholipids regulates exocytosis and gives a new insight into the significance of lipid asymmetry in the plasma membrane.

3T3 Cells↗

PubMed2002

Activation of receptor tyrosine kinases promotes gene transfection in rat neuronal PC12 cells by cationic liposomes.

Cationic liposomes are commonly used to introduce foreign genes to the target cells. However, the methods are not applicable to several types of cells such as neuronal cells. We introduced the luciferase gene to the neuronal pheochromocytoma-12 (PC12) cells by cationic liposomes with a cationic cholesterol derivative (I) or 3- beta - [N - (N',N' - dimethylaminoethane) - carbamoyl]- cholesterol (DC - Chol). Efficiency of gene transfection into undifferentiated naive PC12 cells was extremely higher by the liposomes with the derivative (I) than that by the liposomes with DC - Chol. In addition, when the luciferase gene was transferred into the naive PC12 cells in the presence of nerve growth factor (NGF), the luciferase activity increased much higher than that in the absence of NGF. The transfection efficiency was also promoted by epidermal growth factor in the naive PC12 cells. This high efficiency was maintained in the differentiated PC12 cells obtained by incubating the cells with NGF for 7 days. We found that the transfection efficiency did not depend mainly on the differentiated states of PC12 cells but depended on the activation of receptor tyrosine kinases. It is supposed that the novel liposomes with the derivative (I) will be useful to study the problems in neuron and neuronal tissues for gene transfection.

Animals↗

PubMed2002

Comparative study of transfection efficiency of cationic cholesterols mediated by liposomes-based gene delivery.

To develop the efficient non-viral vector for gene delivery, we compared transfection activities of cationic cholesterol derivatives. We found that the stability of the liposome-DNA complex in the presence of endosome deeply related to the transfection efficiency. We also found that the introduction of a hydrophilic group to the amino terminal of the cholesterol derivative decreased stability and facilitated the release of DNA from the endosome, resulting in higher transfection efficiency.

3T3 Cells↗

PubMed2002

Cationic cholesterol promotes gene transfection using the nuclear localization signal in protamine.

PURPOSE: The purpose of this study was to evaluate protamine-mediated gene transfection by liposomes with a novel cationic cholesterol derivative (I) compared to those with DC-Chol or DOTMA (Lipofectin). METHOD: Plasmid pGL3 DNA was complexed to the cationic liposomes with the derivative (I), DC-Chol, or DOTMA in SFM101(Nissui) at room temperature for 15 min, and thereafter the complex was incubated with target cells (NIH3T3) for 4 h at 37 degrees C. The cells then were washed and cultured for another 40 h in the growth medium at 37 degrees C before luciferase assay. RESULTS: The transfection efficiency by the liposomes with the derivative (I) was much higher than that by the liposomes with DC-Chol or DOTMA. In addition, its transfection efficiency was enhanced greatly by the addition of protamine. Atomic force microscopy showed clearly how the size of the DNA-liposome complex was changed by protamine. Furthermore, fluorescence microscopic images showed that Cy5-labeled antisense DNAs were transferred quicker into the nucleus of the target cells by the liposomes with the derivative I in the presence of protamine. CONCLUSION: Although there exist several possible mechanisms, such as improved protection of DNA intracellularly by derivative (I), one possibility is that the DNA-protamine-liposome complex with the derivative (I) promoted gene transfection more significantly into the nucleus of the target cells using the nuclear localization signal of protamine.

3T3 Cells↗