Biomedical subjects

M Nakashima

Publications and source records attributed to M Nakashima.

At least 109 records · Page 6Linked to original sources

PubMed1999

Milrinone, a phosphodiesterase inhibitor, suppresses intimal thickening after photochemically induced endothelial injury in the mouse femoral artery.

The effect of milrinone, a phosphodiesterase (PDE) inhibitor, on intimal thickening after endothelial denudation was investigated. Intimal thickening was induced in the femoral arteries of mice by a photochemical reaction between rose bengal and transluminal green light which caused endothelial injury followed by platelet adhesion, aggregation, and formation of an occlusive thrombus in the irradiated segment of the mouse femoral artery. In this model, intimal thickening occurred following spontaneous thrombolysis. The intima/media ratio at 21 days after irradiation was 0.556+/-0.104 in the untreated group. Oral administration of milrinone (0.3-3.0 mg/kg) for 3-21 days suppressed intimal thickening by up to 56% in a dose- and time-dependent manner. In an in vivo experiment using bromodeoxyuridine incorporation, milrinone suppressed cell proliferation at 1.0 mg/kg p.o. On the other hand, the minimum doses of milrinone for suppression of ex vivo platelet aggregation induced by collagen (0.8 microg/ml) or ADP (0.5 microM) were 3.0 and 10.0 mg/kg, respectively. These results indicate that milrinone may not suppress intimal thickening by inhibiting platelet function but by preventing vascular smooth muscle cell (VSMC) proliferation, probably through a mechanism mediated via 3', 5'-adenosine cyclic monophosphate (cAMP).

Animals↗

PubMed1999

Inhibitory effect of a new anti-rheumatic drug T-614 on costimulatory molecule expression, cytokine production, and antigen presentation by synovial cells.

The present study was undertaken to investigate the immunoregulatory effects of T-614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne) on synovial cells in vitro. Synovial cells were cultured with T-614 in the presence or absence of various cytokines. After incubation, the costimulatory molecule expression on synovial cells and cytokine production in culture supernatants were analyzed by an indirect immunofluorescence method and enzyme-linked immunosorbent assay, respectively. We also examined the effect of T-614 on the function of synovial cells as antigen-presenting cells (APCs). The costimulatory molecules including CD54, CD58, and CD106 were constitutionally expressed on the surface of synovial cells. However, neither CD80 nor CD86 nor CD102 was found on the surface, and these costimulatory molecules could not be induced by any cytokines. T-614 itself did not affect the costimulatory molecule expression and cytokine production of unstimulated synovial cells. The stimulation of synovial cells with interferon-gamma (IFN-gamma), interleukin-1beta, or 12-O-tetradecanoyl phorbol 13-acetate enhanced the expression of costimulatory molecules and the proinflammatory cytokine production of these cells. Both the up-regulated expression of these costimulatory molecules and the enhanced production of proinflammatory cytokines were significantly inhibited by T-614. Autologous T cell proliferation in response to purified protein derivative by IFN-gamma-treated synovial cells was significantly suppressed by T-614. T-614 has considerable immunosuppressive effects on synovial cells by inhibiting the costimulatory molecule expression and cytokine production of these cells and the antigen-specific T cell proliferation mediated by the synovial cells. These results suggest that T-614 plays an important immunoregulatory role in rheumatoid synovial tissues.

Adjuvants, Immunologic↗

PubMed1999

Prevention of ischemia-induced hippocampal neuronal damage by 2-deoxy-D-glucose in gerbils.

It has been reported that delayed neuronal death (DND) in the hippocampus following transient forebrain ischemia is associated with internucleosomal DNA fragmentation, indicating apoptosis. This suggests that the process of DND is energy dependent. Transient severe forebrain ischemia was induced in Mongolian gerbils by bilateral occlusion of the common carotid arteries. Post-ischemic administration of 2-deoxy-D-glucose (2-DG), a glucose antimetabolite, markedly reduced the occurrence of ischemia-induced DNA fragmentation and DND in the hippocampus. These results suggest that the reduction of energy dependent metabolism after ischemia may be an attractive therapeutic strategy for preserving hippocampal neurons vulnerable to ischemia.

Animals↗

PubMed1999

Effects of dothiepin on delayed conduction produced by ventricular arrhythmia in the canine heart after myocardial infarction.

1. In order to clarify the arrhythmogenic effects of antidepressants, the authors examined the effects of dothiepin and amitriptyline on the ventricular activation time (VAT), effective refractory periods(ERP) and incidence of arrhythmias induced by programmed electrical stimulation(PES) in the dog heart in situ after myocardial infarction. 2. Myocardial infarction was produced by two-stage ligation of the left anterior descending coronary artery. Seven days after ligation, bipolar electrodes were sutured on the ventricular surface of the infarcted and normal zones to apply an electrical stimulation or record ventricular activation. An electrical stimulation with coupling interval 250 or 180 ms was applied on the ventricular surface, and AT was measured. 3. Dothiepin at doses of 1-3 mg/kg increased the heart rate. The VAT of coupling interval 180 ms in the infarcted zone was increased by the administration of 3 mg/kg dosulepin. Dothiepin at 3 mg/kg increased the incidence of ventricular arrhythmias induced by PES. 4. Amitriptyline, at doses of 1-3 mg/kg, significantly increased the heart rate. Amitriptyline increased the VAT dose- and frequency-dependently(2,3 mg/kg zone), and prolonged the ERP and QT c interval. Amitriptyline at doses of 1-3 mg/kg increased the incidence of ventricular arrhythmias by PES. 5. These results indicate that dothiepin, 1-3 mg/kg, has lesser effects on cardiac delayed conduction produced by ventricular arrhythmia than amitriptyline.

Animals↗

PubMed1999

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat.

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.

Animals↗

PubMed1999

Expression of growth/differentiation factor 11, a new member of the BMP/TGFbeta superfamily during mouse embryogenesis.

We have cloned and characterized a new member of the bone morphogenetic protein/transforming growth factor beta (BMP/TGFbeta) superfamily, growth differentiation factor 11 (Gdf11), from rat incisor pulp RNA by reverse transcription-polymerase chain reaction using degenerate primers. The mature carboxyl-terminal domain encoded by Gdf11 is most closely related to Gdf8, being 90% identical to the mouse gene. Northern blot analysis revealed Gdf11 is expressed in adult dental pulp and brain. In situ hybridization of sections and whole-mount embryos demonstrated Gdf11 is first strongly expressed in restricted domains at 8.5 days post coitus (dpc) when it is highest in the tail bud. At 10.5 dpc, it is expressed in the branchial arches, limb bud, tail bud and posterior dorsal neural tube. Later, it is expressed in terminally-differentiated odontoblasts, the nasal epithelium, retina and specific regions of the brain.

Animals↗

PubMed1999

Neuronal apoptosis studied by a sequential TUNEL technique: a method for tract-tracing.

A novel tract-tracing procedure by using a sequential in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling of DNA fragments (TUNEL) is described. This method identifies fragmented DNA transported into neuronal fibers in tissue sections of gerbil hippocampal CA1 neurons following transient forebrain ischemia. The transported DNA has been confirmed by another method, fluorescence DNA detection technique by DAPI. Many methods have been developed to study the neuroanatomical connections in the central nervous system. Principally, these techniques are based on tract-tracing studies using xenobiotics into the central nervous system. Our tract-tracing method is originated from an intrinsic marker that is produced during the apoptotic process of neurons. Furthermore, the advantage of this method is that only the selected cells undergoing apoptosis are recognized and traced to the end of the related neuronal fiber. Usually, apoptotic cells possess intact intracellular metabolic mechanisms until completion of cell death. Thus, apoptotic neurons retain the axonal transport mechanisms which enables us to detect fragmented DNA moving from nuclei to distal terminals of neuronal fibers. Since TUNEL-positive DNA movement within neuronal fibers occurs only during a limited period, it is essential that a time-course of the TUNEL technique is used to study tract-tracing of apoptotic neurons. Although this method can identify only the apical dendrites of cells that are undergoing apoptosis during the limited period, some projections of the gerbil hippocampal CA1 neurons undergoing apoptosis are clearly demonstrated.

Animals↗

PubMed1999

Characterization of ocular pharmacokinetics of beta-blockers using a diffusion model after instillation.

PURPOSE: To characterize the ocular pharmacokinetics of beta-blockers (timolol and tilisolol) after instillation in the albino rabbit using a mathematical model that includes a diffusion process. METHODS: The disposition of fluorescein isothiocyanate-dextran (FITC-dextran, molecular weight 4400), timolol, and tilisolol was determined in tear fluid and aqueous humor after instillation or ocular injection in rabbits. The in vivo penetration parameters were estimated by fitting the concentration-time profiles to the Laplace equations based on a diffusion model using MULTI(FILT) program. The in vitro permeability of drugs was measured across cornea using a two-chamber diffusion cell. RESULTS: Concentration-time profiles of drugs in the tear fluid after instillation showed a monoexponential curve. Although a monoexponential curve was observed in the aqueous humor concentration of FITC-dextran after injection into the aqueous chamber, timolol and tilisolol showed a biexponential curve. On the basis of these results, an in vivo pharmacokinetic model was developed for estimation of penetration parameters. The in vitro partition parameters were higher than those of the in vivo parameters. CONCLUSIONS: The ocular absorption of timolol and tilisolol was characterized using an in vivo pharmacokinetic model and in vivo penetration parameters.

Absorption↗

PubMed1999

Inhibition of cell growth and induction of apoptotic cell death by the human tumor-associated antigen RCAS1.

Tumor-associated antigens that can be recognized by the immune system include the MAGE-family, p53, MUC-1, HER2/neu and p21ras. Despite their expression of these distinct antigens, tumor elimination by the immune system is often inefficient. Postulated mechanisms include insufficient expression of co-stimulatory or adhesion molecules by tumor cells, or defective processing and presentation of antigens on their cell surfaces. Tumor cells may also evade immune attack by expressing CD95 (APO-1/Fas) ligand or other molecules that induce apoptosis in activated T cells. Here we describe RCAS1 (receptor-binding cancer antigen expressed on SiSo cells), a membrane molecule expressed on human cancer cells. RCAS1 acts as a ligand for a putative receptor present on various human cell lines and normal peripheral lymphocytes such as T, B and NK cells. The receptor expression was enhanced by activation of the lymphocytes. RCAS1 inhibited the in vitro growth of receptor-expressing cells and induced apoptotic cell death. Given these results, tumor cells may evade immune surveillance by expression of RCAS1, which would suppress clonal expansion and induce apoptosis in RCAS1 receptor-positive immune cells.

Amino Acid Sequence↗

PubMed1999

Pituitary tumor transforming gene (PTTG) expression in pituitary adenomas.

We recently cloned a novel pituitary tumor transforming gene (PTTG). Here we report PTTG expression in human pituitary adenomas and in normal pituitary tissue. In situ hybridization revealed PTTG expression in nonfunctioning and in GH-secreting adenomas but not in normal pituitary tissue. Using a more sensitive detection method, RT-PCR, low level PTTG expression was detected in normal pituitary. However, when expression levels in normal pituitary tissue were compared with those in 54 pituitary tumors using comparative reverse transcription polymerase chain reaction (RT-PCR), we found that most tumor samples expressed higher levels of PTTG. More than 50% PTTG increases were observed in 23 of 30 nonfunctioning pituitary tumors, all 13 GH-producing tumors, 9 of 10 prolactinomas, and 1 ACTH-secreting tumor, with more than 10-fold increases evident in some tumors. Furthermore, higher PTTG expression (P = 0.03) was observed in hormone-secreting tumors that had invaded the sphenoid bone (stages III and IV; 95% CI 3.118-9.715) compared with hormone-secreting tumors that were confined to the pituitary fossa (stages I and II; 95% CI 1.681-3.051). Therefore, PTTG abundance is a molecular marker for invasiveness in hormone-secreting pituitary tumors. The ubiquitous and prevalent expression of pituitary adenoma PTTG suggests that PTTG plays a role in pituitary tumorigenesis and invasiveness.

Adenoma↗

PubMed1999

Structure, expression, and function of human pituitary tumor-transforming gene (PTTG).

Despite advances in characterizing the pathophysiology and genetics of pituitary tumors, molecular mechanisms of their pathogenesis are poorly understood. Recently, we isolated a transforming gene [pituitary tumor-transforming gene (PTTG)] from rat pituitary tumor cells. Here we describe the cloning of human PTTG, which is located on chromosome 5q33 and shares striking sequence homology with its rat counterpart. Northern analysis revealed PTTG expression in normal adult testis, thymus, colon, small intestine, brain, lung, and fetal liver, but most abundant levels of PTTG mRNA were observed in several carcinoma cell lines. Stable transfection of NIH 3T3 cells with human PTTG cDNA caused anchorage-independent transformation in vitro and induced in vivo tumor formation when transfectants were injected into athymic mice. Overexpression of PTTG in transfected NIH 3T3 cells also stimulated expression and secretion of basic fibroblast growth factor, a human pituitary tumor growth-regulating factor. A proline-rich region, which contains two PXXP motifs for the SH3 domain-binding site, was detected in the PTTG protein sequence. When these proline residues were changed by site-directed mutagenesis, PTTG in vitro transforming and in vivo tumor-inducing activity, as well as stimulation of basic fibroblast growth factor, was abrogated. These results indicate that human PTTG, a novel oncogene, may function through SH3-mediated signal transduction pathways and activation of growth factor(s).

3T3 Cells↗

PubMed1999

Construction of the single-chain Fv from 196-14 antibody toward ovarian cancer-associated antigen CA125.

The variable regions of heavy- and light-chains of mouse monoclonal antibody 196-14 toward ovarian cancer-associated antigen CA125 were linked with a peptide linker (GSTSGSGKSSEGKG) and a histidine tag was attached at the carboxyl terminal. This single-chain Fv (scFv) with a histidine tag was expressed in Escherichia coli as an inclusion body. The inclusion body was solubilized with guanidium chloride, followed by purification on nickel nitrilotriacetic acid agarose column and refolding into the active form. The scFv thus obtained bound to the Siso cells, which express CA125, and may recognize the same epitope as the parental 196-14 antibody IgG does.

Animals↗

PubMed1999

Characterization of ocular pharmacokinetics of tilisolol after instillation into anesthetized rabbits.

The purpose of this study was to characterize the ocular pharmacokinetics of a beta-blocker, tilisolol, after instillation into anesthetized rabbits using a mathematical model including a diffusion process. The samples were analyzed by HPLC. Anesthetized rabbit was used as a model of tear secretion deficiency. Anesthetized rabbits showed higher drug concentration in the tear fluid and aqueous humor after instillation than unanesthetized rabbits. A mathematical model including a diffusion process and in vivo penetration parameters well described the concentrations of tilisolol in the aqueous humor after instillation in anesthetized rabbits.

Administration, Topical↗

PubMed1999

A highly sensitive ELISA for the quantification of polymyxin B sulfate in human serum.

A highly sensitive ELISA for the determination of polymyxin B sulfate (PMB) was developed which is capable of measuring as low as 32 pg/ml. Anti-PMB antibody was obtained by immunizing rabbits with PMB conjugated with mercaptosuccinyl bovine serum albumin (MS. BSA) using N-(gamma-maleimidobutyryloxy) succinimide (GMBS) as a heterobifunctional coupling agent. An enzyme marker was similarly prepared by coupling PMB with horseradish peroxidase (HRP) employing GMBS. This ELISA showed very low reactivity with the PMB analogue, polymyxin E (0.05%). The values for PMB concentration detected by this assay were comparable with those detected by the bioassay. Moreover, the ELISA was about 10,000 times more sensitive in detecting PMB at lower concentrations. Serum PMB concentration after the oral administration of a PMB tablet to human subjects was determined by the ELISA. PMB was rapidly absorbed from the gastrointestinal tract after the administration, then slowly decreased. These results indicate that the ELISA may be a valuable tool for studies of the pharmacokinetics and pharmacodynamics of the anti-endotoxin drug, PMB.

Administration, Oral↗

PubMed1999

In vitro characteristics and in vivo plasma disposition of cisplatin conjugated with oxidized and dicarboxymethylated dextrans.

In vitro release behavior and cytotoxic activity, and in vivo plasma disposition of newly synthesized macromolecular derivatives of cisplatin (CDDP) were investigated and compared with CDDP. The derivatives included oxidized dextran conjugate of CDDP (OX-Dex/CDDP) and dicarboxymethylated dextran conjugate of CDDP (DCM-Dex/CDDP). In vitro release of platinum complex from dextran conjugated CDDP was determined by an equilibrium dialysis method. These dextran conjugates showed sustained release of the platinum complex. In vitro release half-life for DCM-Dex/CDDP was significantly longer (4.5 times) than that for OX-Dex/CDDP. In vitro cytotoxic activity of CDDP and dextran conjugated CDDP against colon 26, mouse colon cancer cell line, was measured using the MTT assay method. OX-Dex/CDDP showed a similar cytotoxic activity to CDDP. However, both cytotoxic activities were markedly decreased when preincubated with the medium containing serum. On the other hand, DCM-Dex/CDDP retained residual cytotoxic activity at a significantly higher level than OX-Dex/CDDP after preincubation with the medium containing serum, although it showed the lowest cytotoxic activity. This indicated longer maintenance of the in vitro antitumor activity of DCM-Dex/CDDP in serum compared with OX-Dex/CDDP. Plasma disposition of CDDP and dextran conjugated CDDP was determined by intravenous administration to rats. Although the total platinum plasma concentration-time profile for OX-Dex/CDDP was similar to that for CDDP, its markedly higher profile was achieved when DCM-Dex/CDDP was administered. The values of the total platinum AUC and MRT, where AUC is the area under the platinum concentration-time curve and MRT is the mean residence time, for DCM-Dex/CDDP were 11.2 times and 4.8 times significantly higher than with OX-Dex/CDDP in plasma, respectively. DCM-Dex/CDDP also showed a significantly lower total clearance compared with OX-Dex/CDDP. These results from the in vivo experiments revealed that retention of DCM-Dex/CDDP in blood circulation was much greater than that for OX-Dex/CDDP. DCM-Dex/CDDP thus has potential as a macromolecular derivative of CDDP for passive tumor targeting.

Animals↗

PubMed1999

Drug absorption behavior after periocular injections.

The purpose of this study was to investigate the absorption behavior of an ophthalmic drug injected in rabbit periocular tissues. After intracapsular, retrobulbar and palpebral conjunctival injections of 150 microl and 50 microl fluorescein isothiocyanate dextran (FITC-dextran, average molecular weight 11000), leakage of the dye into the tear fluid was dependent on the injection route and volume. After periocular injections (50 microl) of tilisolol, as a model beta-blocker, the concentrations in the tear fluid, blood, aqueous humor and vitreous body were determined by HPLC. Slight drug leakage was observed in the tear fluid after injections. The periocular injections showed a faster absorption and a higher area under the concentration-time curve (AUC) in the plasma and a lower AUC in the aqueous humor than those observed in instillation. They also showed a higher ratio of AUC of tilisolol in the vitreous body to AUC in the aqueous humor than that observed in the instillation. Among the periocular injections, retrobulbar injection showed the highest concentrations in the plasma and the lowest in the aqueous humor and vitreous body, while intracapsular injection showed the lowest in the plasma and the highest in the aqueous humor and vitreous body. Although the periocular injections showed a rapid systemic absorption of drug by a rich topical vasculature, it might be an effective approach to deliver the drug to the periocular tissues and vitreous body.

Absorption↗

PubMed1999

Topical delivery system of ophthalmic drugs by periocular injection with viscous solution.

The purpose of this study is to evaluate periocular injections with viscous solution as a topical delivery system of ophthalmic drugs. Tilisolol and carboxymethylcellulose (CMC) were used as a model beta-blocker and a viscous polymer, respectively. After intracapsular, retrobulbar and palpebral conjunctival injections (50 microl) of tilisolol with 3% CMC into rabbits, drug concentrations in the tear fluid, blood, aqueous humor and vitreous body were determined by HPLC. Periocular injection (50 microl) of tilisolol with 3% CMC showed slight leakage of the drug in the tear fluid from the injection site. The viscous vehicle decreased the absorption rate constant of the drug from the injection site to systemic circulation compared with the buffer solution. It suggests that the viscous solution improved the retention of drug at both the injection site and in periocular tissues. Although the periocular injections with viscous vehicle (3% CMC) showed lower AUC in the aqueous humor than that observed in instillation, they showed comparable AUC in the vitreous humor. Compared to the results after the periocular injections with buffer solution, CMC increased the AUCs in the vitreous body 3.1-fold with retrobulbar injection and 1.4-fold with palpebral conjunctival injection, respectively. As a result, periocular injections with 3% CMC showed higher delivery of tilisolol to the vitreous body against the aqueous humor than the instillation and periocular injections with buffer solution.

Absorption↗

PubMed1999

[Clinicopathological analysis of transition zone cancer of the prostate: the clinical significance of coexistent non-transition zone cancer foci].

BACKGROUND: Dominant cancer of transition zone origin of the prostate (TZ cancer) has been frequently detected, because ultrasound-guided systematic biopsies have been generalized. In cases of TZ cancer, we attempted to determine clinical significance of coexistent non-TZ cancer foci. MATERIALS AND METHODS: Twenty cases with TZ cancer who underwent radical prostatectomy or cystoprostatectomy were clinicopathologically evaluated using step-sectioned specimens. RESULTS: In TZ cancer foci, there were extraprostatic extension in 5 cases (25%), seminal vesicle invasion in 2 cases (10%), positive surgical margin in 6 cases (30%) and bladder neck invasion in 4 cases (20%). The extraprostatic extension and the positive surgical margin occurred at the anterior or anterioapical portion of the prostate in all the cases. On the other hand, 17 (85%) had coexistent non-TZ cancer foci. In non-TZ cancer foci, there were extraprostatic extension in 3 cases (15%), seminal vesicle invasion in 1 case (5%) and positive surgical margin in 1 case (5%). The extraprostatic extension and the positive surgical margin occurred at the posteriolateral portion of the prostate in all the cases. In 3 cases a coexistent non-TZ cancer focus showed the extraprostatic extension, the seminal vesicle invasion or the positive surgical margin, although a TZ cancer focus were organ-confined. CONCLUSION: We should add attention to coexistent non-TZ cancer foci in TZ cancer cases. Particularly, we believe that pre-operative evaluation of non-TZ cancer foci is needed in TZ cancer cases of the candidates for nerve-sparing radical prostatectomy.

Aged↗