Biomedical subjects

T Takizawa

Publications and source records attributed to T Takizawa.

At least 19 recordsLinked to original sources

PubMed1999

Glucose-6-phosphatase is present in normal and pre-eclamptic placental trophoblasts: ultrastructural enzyme-histochemical evidence.

The purpose of the present study was to localize glucose-6-phosphatase (G6Pase) activity in the human placenta at various gestational stages and to compare them to pre-eclamptic placenta activity. Ultrastructural enzyme-histochemical analysis of G6Pase was performed using cerium and lead as capturing agents. Precipitates indicative of G6Pase activity were observed in the endoplasmic reticulum and the nuclear envelope of the syncytiotrophoblasts in near-term placenta obtained from women with normal pregnancies. In placenta taken from women with severe pre-eclampsia, the localization pattern, enzyme activity intensity, and morphology of the endoplasmic reticulum did not differ from normal pregnancies. Stringent control experiments were performed also to ensure specific detection of G6Pase activity. The results indicate that cytochemically detectable G6Pase is present in the human placenta. This enzyme may play significant roles in carbohydrate metabolism in the human placenta.

Endoplasmic Reticulum↗

PubMed1999

Double-stranded RNA-activated protein kinase (PKR) fused to green fluorescent protein induces apoptosis of human embryonic kidney cells: possible role in the Fas signaling pathway.

PKR is an interferon-inducible, double-stranded (ds) RNA-activated serine/threonine protein kinase, and has been shown to play roles in viral pathogenesis, cell growth and apoptosis. We expressed PKR as a fusion protein with enhanced jellyfish green fluorescence protein (EGFP) in human embryonic kidney 293 cells to visualize the effect of PKR transfection. The EGFP-fusion construct with wild-type PKR showed both auto- and substrate-phosphorylation activities independent of dsRNA, indicating EGFP-PKR is constitutively active. The EGFP-construct with a mutant PKR with the first RNA binding domain deleted still possessed kinase activities. On the other hand, the EGFP-fusion with a catalytically inactive mutant of PKR with the substitution of K at 296 with R, which has been shown to have tumorigenic properties, did not possess kinase activities. Transfection of the constitutive active forms of EGFP-PKR constructs induced apoptosis in 293 cells without dsRNA, whereas the EGFP-fusion with the catalytically inactive mutant did not cause apoptosis but rather protected cells from Fas-induced cell death. In addition, Fas-stimulation increased endogenous PKR activities. These results constitute evidence that PKR is sufficient to induce apoptosis, and plays a role in Fas-mediated apoptosis.

Animals↗

PubMed1998

Ultrasmall immunogold particles: important probes for immunocytochemistry.

In this article, we review the immunocytochemical literature with respect to a comparison between conventional colloidal gold and ultrasmall gold particles as immunoprobes. We discuss the relative advantages and disadvantages of each of these types of particles for immunocytochemical applications. We present results from our own laboratories, in which we compared these immunoprobes in selected experimental situations. In addition, we discuss our work on the use of a fluorescently labeled ultrasmall immunoprobe for correlative microscopy.

Fluorescent Dyes↗

PubMed1998

Ultrastructure of human scalp hair shafts as revealed by freeze-substitution fixation.

Human scalp hair is important as a diagnostic clue to many diseases, in medical jurisprudential investigations, and also as a subject of cosmetic treatments. While many ultrastructural studies of the human hair root including the hair follicle have been reported, few studies have been done on the human hair shaft. We report here the ultrastructure of human scalp hair shafts prepared by a rapid-freezing technique followed by freeze-substitution fixation that allows the observation of fine cell structures. Healthy scalp hair shafts from Japanese females 12-13 years of age were rapid-frozen and then freeze-substituted in OsO4-acetone. In addition, this technique was applied to the study of some changes of the hair shafts (i.e., hair damaged by thioglycolic acid cold permanent waving and white hair). By this method, the hair shaft was rapid-frozen throughout without appreciable ice damage although the hair shaft was nearly 100 microm in diameter. The rapid-freezing technique resulted in excellent preservation of the ultrastructure of the hair shafts: lamellar structures in the cuticle and fine fibrous ultrastructures in the cortex were observed without chemical treatments. Thioglycolic acid treatment affected the ultrastructure of both the cuticle and the cortex. Except for the absence of melanin granules, no significant differences in the ultrastructure were observed between white hair and black hair. The rapid-freezing technique followed by freeze-substitution fixation appears to be the most reliable approach for the morphological evaluation of fully keratinized cells and tissues.

Adolescent↗

PubMed1998

Sarcoplasmic reticulum genes are selectively down-regulated in cardiomyopathy produced by doxorubicin in rabbits.

The clinical utility of doxorubicin, an antineoplastic agent, is limited by its cardiotoxicity. Our objective was to determine whether expression of genes encoding proteins that affect Ca2+ homeostasis were altered in the hearts of rabbits chronically treated with doxorubicin. Twelve male New Zealand white rabbits received an injection of doxorubicin (2.5 mg/kg i.v.) once a week for 8 weeks. Eight rabbits were similarly injected with saline as controls. The cardiac function of both groups was evaluated 8 weeks after the final injection, as were the levels of expression of mRNA for Ca2+ transport proteins in the sarcoplasmic reticulum and plasma membrane. The amount of the sarcoplasmic reticulum Ca2+-ATPase and the Ca2+ uptake capacity of the protein were also quantitated. Cardiac output was significantly decreased in the doxorubicin-treated group (71+/-21 ml/min, P<0.05) compared with the control group (118+/-15 ml/min). The mRNA levels for the sarcoplasmic reticulum proteins were significantly diminished in the doxorubicin-treated hearts: ryanodine receptor-2 (relative expression level compared with controls, 0.35+/-0.13, P<0.01), sarcoplasmic reticulum Ca2+-ATPase (0.56+/-0.13, P<0.01), phospholamban (0.62+/-0.20, P<0.01) and cardiac calsequestrin (0. 57+/-0.26, P<0.01). In addition, both relative amount of sarcoplasmic reticulum Ca2+-ATPase protein (doxorubicin-treated group, 69+/-17% of control, P<0.01) and the Ca2+ uptake capacity (46. 9+/-9.8 nmol Ca2+/mg protein-5 min in doxorubicin group v 63.2+/-10. 4 in the control group, P<0.01) were concomitantly decreased with its mRNA expression level. Conversely, the mRNA levels for the plasma membrane proteins did not differ from those of control rabbits: the dihydropyridine receptor (relative expression level, 1. 03+/-0.30, N.S.), plasma membrane Ca2+-ATPase (0.93+/-0.33, N.S.) and the Na+/Ca2+ exchanger (0.87+/-0.34, N.S.). These findings suggest that a selective decrease in mRNA expression for sarcoplasmic reticulum Ca2+ transport proteins is responsible for the impaired Ca2+ handling, and thus, for the reduced cardiac function seen in the cardiomyopathy induced in rabbits by the long-term treatment with doxorubicin.

Animals↗

PubMed1998

Efficient gene transfer into cardiac myocytes using adeno-associated virus (AAV) vectors.

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing beta-galactosidase (beta-gal) gene in vitro, and the expression of beta-gal in CM was evaluated by X-gal staining and beta-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the beta-gal expression increased to 31.1 +/- 4.6 ng/mg protein in a MOI-dependent manner (MOI: 10(4) to 10(6) particles/cell). The beta-gal expression was also increased in an incubation period-dependent manner (1-24 h). beta-gal expression was maximal at day 3 and then gradually decreased with time. However, beta-gal expression at day 14 was almost the same level as that at day 1 (45.5 +/- 5.9 v 55.2 +/- 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the beta-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.

Animals↗

PubMed1998

Interaction of gangliosides with proteins depending on oligosaccharide chain and protein surface modification.

By using neutron and synchrotron x-ray small-angle scattering techniques, we investigated the process of the complexation of gangliosides with proteins. We treated monosialoganglioside (G(M1)), disialoganglioside (G(D1a)), and a mixture of G(M1)/G(D1a). Proteins used were bovine serum albumins whose surfaces were modified with different sugars (deoxy-D-galactose, deoxy-L-fucose, deoxymaltitol, and deoxycellobiitol), which were used as model glycoproteins in a membrane. We found that the complexation of gangliosides with albumins greatly depends on the combination of ganglioside species and protein surface modification. With a varying protein/ganglioside ratio in a buffer solution at pH 7, the complexation of G(M1) or G(D1a) with albumins modified by monosaccharides appears to be less destructive for ganglioside aggregate structures in forming large complexes; the complexation of G(D1a) with the albumins modified by disaccharides induces the formation of complexes with a dimeric structure; and the complexation of G(M1) with albumins modified by disaccharides, to form small complexes, is very destructive. The present results show a strong dependence of the interaction between ganglioside and protein on the characteristics of the ganglioside and protein surface, which would relate to a physiological function of gangliosides, such as a function regulating the receptor activity of glycoproteins in a cell membrane.

Animals↗

PubMed1998

Intensive extrusion and occlusion of water in ganglioside micelles with thermal reversibility.

By using a shell-modeling analysis for small-angle scattering data of ganglioside micellar dispersion, we recently reported that the elevation of temperature induces a significant shrinkage of the hydrophilic region of the ganglioside micelle, suggesting that the oligosaccharide chains with sialic acid residues of ganglioside molecules change the conformation, sensitively responding to a change in temperature (Hirai et al., 1996. Biophys. J. 70:1761-1768; J. Phys. Chem. 100:11675-11680). We have carried out further analyses of the temperature dependence of the structural parameters reported previously, and we have found clear evidence of reversible extrusion and occlusion of a large amount of water in the hydrophilic region of the ganglioside micelle in the physiological temperature range of 6-60 degrees C. The present results suggest a remarkable function of ganglioside molecules: they change the hydrophilicity of the cell surface locally as a response to variations in temperature. This phenomenon might be involved in various surface events, such as cell-cell interaction and cell surface-protein interaction.

Animals↗

PubMed1998

Lymph node metastasis in small peripheral adenocarcinoma of the lung.

OBJECTIVE: Our aim in this study is to clarify the clinical and pathologic features of small peripheral adenocarcinoma of the lung with special emphasis on intraoperative identification of lymph node metastasis. PATIENTS AND METHODS: Between 1980 and 1996, 157 patients underwent lobectomy and complete hilar/mediastinal lymphadenectomy for small (1.1 to 2.0 cm in diameter) peripheral adenocarcinoma of the lung. The intraoperative assessment, the distribution of metastatic lymph nodes, and the association between the tumor's histopathologic characteristics and lymph node metastasis were retrospectively investigated in this study. RESULTS: Postoperative examination revealed lymph node metastasis in 27 (17%) patients. Lymph node metastases were not noticed during the operation in 19 of these 27 patients. Metastases were localized in single lymph nodes in 10 patients; the metastases were distributed over a segmental, a lobar, an interlobar, and a mediastinal lymph node. The prevalence of lymph node metastasis was as follows: Of 92 patients with well-differentiated adenocarcinoma, seven (8%) had lymph node metastases; of the 65 patients with other types of tumors, 20 (31%) had lymph node metastases. Of 120 patients without pleural involvement, 13 (11%) had lymph node metastases; of the 37 with pleural involvement, 14 (38%) had lymph node metastases. Five-year survivals were estimated at 91% +/- 6% (mean +/- 95% confidence interval) for 130 patients with N0 tumor and 30% +/- 22% for 27 patients with N1 or N2 tumor. CONCLUSIONS: Intraoperative assessment is not reliable for identifying lymph node metastasis. Lobectomy and complete hilar/ mediastinal lymphadenectomy are necessary to determine N stage rigidly. Histologic degree of differentiation and pleural involvement are significantly associated with lymph node metastasis.

Adenocarcinoma↗

PubMed1998

Clinical analysis of small-sized peripheral lung cancer.

OBJECTIVE: In Japan, with the initiation of the lung cancer screening program, small-sized peripheral lung cancer in which the diameter is 2 cm or less has been increasing. The purpose of this study is to determine the clinicopathologic behavior of small-sized lung cancer. METHODS: Four hundred ninety-six patients with cT1 N0, peripheral, resected non-small-cell lung cancer, who were operated on between 1980 and 1996, were selected, grouped by tumor diameter or histologic type, and then analyzed for clinicopathologic behavior. On the basis of measured diameter roentgenographically, the patients were divided into two groups; group c-S with lesions 2 cm or less in diameter and group c-L with lesions 2.1 to 3 cm in diameter. RESULTS: Lymph node metastasis was recognized in 18% of group c-S, in 23% of group c-L, and in 21% for the entire clinical group. The rate of those with the progressive state was 19% in group c-S and 26% in group c-L. The 5-year survival was 79.5% in group c-S and 69.3% in group c-L (i.e., there was a significant difference between the two groups). CONCLUSION: Compared with the patients with lesions 2.1 to 3 cm in diameter, the patients with small-sized lung cancer had a milder progressive state and a better prognosis.

Adult↗

PubMed1998

A resected case of hilar type double primary lung cancer following endobronchial brachytherapy.

A 61-year-old man with squamous cell carcinoma of the right B1 and the left second carina which extended to the left main bronchus, was treated with low dose rate brachytherapy bilaterally prior to resection. A complete response was gained at the right B1 and the left main bronchus, a resection of the left apical segment and the second carina was carried out with bronchoplastic procedures. The patient remains disease-free 2 years after the operation and maintains a good quality of life. In this case, the preoperative treatment with brachytherapy was effective.

Brachytherapy↗

PubMed1998

Foreign body gonitis caused by a broken poly-L-lactic acid screw.

Poly-L-lactic acid (PLLA), a biodegradable polymer, is generally considered to be gradually degraded without causing any severe tissue reaction. However, we encountered a patient who developed foreign body gonitis caused by screw breakage after fixation of an intercondylar prominence fracture with PLLA screws. This case suggests that care should be taken when PLLA materials are used to fix intraarticular fractures.

Adult↗

PubMed1998

Intravascular ultrasound imaging of atherosclerotic renal arteries: comparison between in vitro and in vivo studies.

BACKGROUND: Intravascular ultrasound (IVUS) imaging, a new modality, may be feasible and useful for the assessment of atherosclerotic renal arteries. However, comparison between in vivo and in vitro studies to confirm pathological changes corresponding with IVUS findings obtained from renal arteries was not fully evaluated. METHODS: We evaluated ultrasound images of 18 post-mortem human renal arteries and cross-sectional IVUS images of main renal arteries in five patients with renal artery stenosis (RAS) or essential hypertension. RESULTS: In vitro studies have shown that renal-artery images had three layers when the arteries had fibrous intimal thickening and medial hypertrophy. Renal arteries, in which the fibrous intima was not well developed, showed circumferentially homogeneous bright echoes. In patients with atherosclerotic RAS and essential hypertension, IVUS images showed hyperechoic areas in the renal arterial walls, probably due to atherosclerosis. Typical three-layered ultrasound appearance was not easily seen during in vivo studies. CONCLUSION: Our findings suggest that hyperechoic images can be a diagnostic clue of atherosclerosis However, in vitro results do not always correspond exactly to in vivo findings, and caution is needed when findings from in vitro IVUS imaging studies are applied to in vivo studies.

Adolescent↗

PubMed1998

Light-induced cGMP-phosphodiesterase activity in intact rat retinal rod cells as revealed by rapid-freezing enzyme cytochemistry.

Cyclic guanosine monophosphate (cGMP)-phosphodiesterase (PDE) in the outer segment of vertebrate retinal rod cells is one of key enzymes mediating phototransduction. We report here on light-induced PDE activity in intact rat retinal rod cells processed by rapid-freezing enzyme cytochemistry, a new morphological technique that is a combination of rapid-freezing, freeze-substitution fixation, and subsequent enzyme cytochemistry. This technique quickly immobilizes and preserves both enzyme activity and the cell ultrastructure in a state approximating living conditions; consequently, it has proved useful for cytochemical detection of light-induced PDE activity. Using this technique we observed that the catalytic site of PDE molecules in rapid-frozen outer segments was predominantly located in the extradiscal spaces; PDE activity was significantly greater in light than in darkness; and illumination elicited marked increases in PDE activity in dark-adapted cells. Light-induced PDE activity was first cytochemically detected after 3 s of illumination and reached a peak within 5 s, at which time it was in virtually the same as that seen in fully light adapted cells. In addition, cytochemical guanosine triphosphatase (GTPase) activity in dark-adapted cells, as well as corresponding PDE activity, increased in a time-dependent manner with illumination duration; this acceleration in GTPase activity closely paralleled the PDE activity. Thus, our results suggest, in part, the existence of reciprocal regulation of PDE and activated transducin alpha subunit, thereby modulating light adaptation in rod cells.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1998

Long-circulating immunoliposomal amphotericin B against invasive pulmonary aspergillosis in mice.

We investigated the efficacy of long-circulating immunoliposomal amphotericin B (AmB) against invasive pulmonary aspergillosis in mice using three types of liposomal AmB: conventional liposomal AmB (AmBisome), a long-circulating liposomal AmB and prepared by coating the liposome surface with polyethylene glycol (PEG; PEG-L-AmB), long-circulating immunoliposomal AmB (34A-PEG-L-AmB). The survival rates for mice with invasive pulmonary aspergillosis treated with an intravenous dose of 2 mg of AmBisome, PEG-L-AmB, or 34A-PEG-L-AmB per kg of body weight were 16.7, 83.3, and 100%, respectively. Treatment with 34A-PEG-L-AmB produced a marked reduction in the number of Aspergillus fumigatus organisms in the lungs. Pharmacokinetic studies showed the presence of high AmB concentrations in the plasma of mice treated with PEG-L-AmB (40.8 microg/ml) and in the lungs of mice treated with 34A-PEG-L-AmB (42.3 microg/g). We conclude that 34A-PEG-L-AmB, a long-circulating immunoliposomal AmB, is a promising form of AmB against invasive pulmonary aspergillosis.

Amphotericin B↗

PubMed1998

5'-nucleotidase in rat photoreceptor cells and pigment epithelial cells processed by rapid-freezing enzyme cytochemistry.

This report describes the subcellular distribution of 5'-nucleotidase (5'-NT) in rat photoreceptor cells and pigment epithelial cells processed by rapid-freeze enzyme cytochemistry. There was a striking difference in the ultrastructural localization of 5'-NT activity between rod outer segments after freeze-substitution fixation and conventional fixation. By rapid-freezing enzyme cytochemistry, 5'-NT activity was localized in the extradiscal space of intact nonvacuolated discs, whereas by conventional cytochemistry it was shown in the intradiscal space of artifactual vacuolated discs. In the freeze-substituted retinal cells, an appreciable difference in functional 5'-NT molecules was also found. The soluble 5'-NT on the cytoplasmic side of the disc membrane was vital in the rod outer segments, whereas the membrane-bound ecto-5'-NT on the exoplasmic (external) surface of the apical process was active in the pigment epithelial cells. Rapid-freezing enzyme cytochemistry should be worth employing as a method to reveal the fine localization of enzyme activity at the level of cell ultrastructures, which are poorly preserved by conventional fixation, and should provide information approximate to that in living cells.

5'-Nucleotidase↗

PubMed1998

Correlative microscopy using FluoroNanogold on ultrathin cryosections. Proof of principle.

We demonstrate a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to be a versatile reporter system for immunocytochemical labeling of ultrathin cryosections. FNG-labeled molecules in the same ultrathin cryosections can be resolved by two imaging techniques (i.e., fluorescence and electron microscopy). Lactoferrin, a marker protein for the specific granules in human neutrophils, was employed as the target for FNG immunolabeling. The spatial resolution of the fluorescence signal from FNG-labeled specific granules was compatible with that of silver-enhanced gold signal from the same granules in electron microscopy. Our results confirm that FNG can be used as a probe for high-resolution correlation between immunofluorescence and electron microscopy.

Frozen Sections↗

PubMed1998

[Construction and its characteristic on the concept of a 'health culture'].

Toward the 21st century, subjects and methods of hygiene and public health will be specialized and subdivided. However, practical approaches to human health need an integrated method focusing to a structure of human life. Under these circumstances, the concept of 'health culture' becomes prevalent. The role of hygiene related to the improvement of life style, the development of re-cycle system, and fulfillment of a barrier-free system focussed on the handicapped and the elderly have become increasingly important. Therefore, an increase in the recognition to the concept of 'health culture' is essential to the research of hygiene. The purpose of this paper is to make a historical and theoretical analysis of health culture', in order to promote it as the leading concept of all activities concerned with health in the 21st century. The methods of this paper are mainly historical and theoretical review. 'Health culture' was introduced in American and European societies a hundred years ago. Health culture in the USA involves knowledge and skills applied to actual daily life, refrecting pragmatism as the ideal feature of American society. In Germany, the concept of 'Hygienishe Kultur' was established at the field of social hygiene by Grotjahn and by Fischer in the early 20th century. This concept recognized the importance of the development of culture and independence of life in labour based on the evolution of the concept of 'Hygienishe Kultur'. In Japan, under the influence of German social hygiene, the social hygienic theory flourished. A social hygienist, Tetsuo Hoshino used the term 'hygienic culture' in the context of life creation toward a healthy life. Health culture is the total system concerning knowledge, experience, skill, and norms related to health, which has developed with the development of society. It has fundamental function promotes the creation of culture and self-cultivation of living man, whereas, it contributes to the realization of health based on individuality, in conjunction with the co-operation of medical and health sciences. The contemporary representation of health culture includes new health care activities such as self-care, a self-help movement, and health volunteer activities. It means the basic shift of the function of health culture is from that of life style to life movement. Nowadays, the role of hygiene in the total health care system is seriously considered. The goal, objects, and methods of hygiene should be re-evaluated under the concept of 'health culture', in order to re-define hygiene as the science for people living in a society.

Attitude to Health↗