Biomedical subjects

Y Uchiyama

Publications and source records attributed to Y Uchiyama.

At least 91 records · Page 5Linked to original sources

PubMed1999

The induction of autophagic vacuoles and the unique endocytic compartments, C-shaped multivesicular bodies, in GH4C1 cells after treatment with 17beta-estradiol, insulin and EGF.

The mechanisms for the formation of autophagic vacuoles were investigated using GH4C1 cells, a rat pituitary tumor cell line, whose induction increases intracellular levels of lysosomal proteinases and their mRNA by treatment with a combination of hormones (17beta-estradiol, insulin and EGF). By ordinary electron microscopy, autophagic vacuoles containing various undigested structures with or without limiting membranes were abundant in the hormone-induced cells. These vacuoles, also containing numerous small vesicles, appeared to be derived from multivesicular bodies. In fact, there were also numerous C-shaped multivesicular bodies which enclosed cytoplasmic portions, suggesting that these unique structures are involved in the production of the autophagic vacuoles. Moreover, the cytoplasmic portions enlapped by the C-shaped multivesicular bodies were high in electron density and contained filamentous structures. By the cryothin-section immunogold method, the C-shaped multivesicular bodies in some cases contained lysosomal marker proteins such as cathepsins B and H, and Igp 120. Using an anti-actin monoclonal antibody, immunogold particles clearly labeled the cytoplasmic portions enclosed by the C-shaped multivesicular bodies. Pulse-chase experiments with horse radish peroxidase, a fluid-phase endocytic marker, revealed that the incidence of the C-shaped multivesicular bodies labeled with horse radish peroxidase peaked at 30 min after the beginning of chase incubation, whereas no C-shaped multivesicular body with horse radish peroxidase was detected in the cells by cytochalasin D treatment. These results suggest that the C-shaped multivesicular bodies occur in a transitional process from endosomes to lysosomes by the action of actin filaments, and that this morphological change may be essential for the production of autophagic vacuoles in the hormone-induced GH4C1 cells.

Actins↗

PubMed1999

[A sensitive enzyme immunoassay for the measurement of small quantities of erythrocyte-associated IgG in patients with systemic lupus erythematosus who had negative direct antiglobulin test].

To measure the amount of erythrocyte-associated IgG (EAIgG) molecules from 70 patients with systemic lupus erythematosus (SLE) who had negative direct antiglobulin test (DAT), we employed a sensitive enzyme-linked immunosorbent assay (ELISA) technique. Ninety-five percent of healthy individuals were less than 65 molecules of EAIgG per red cell (RBC). About 51 percent of these patients with SLE were positive (over 65 molecules of EAIgG per RBC) by this technique that showed more sensitive than the DAT. EAIgG levels of these patients were inversely proportional to RBC count (r = -0.369, p < 0.005), and EAIgG positive patients were recognized by 88 percent in patients group who were less than 4 x 10(6)/microliter RBC counts. These results suggest that even a few EAIgG, as of a DAT shows negative, are related to the catabolism of erythrocytes in patients with SLE. EAIgG determination value in this method calculated and converted with CRM 470 that is the IFCC international reference preparation for plasma protein.

Biomarkers↗

PubMed1999

[Surgical treatment of lung cancer with combined resection of the aorta or superior vena cava].

Until December 1997, 699 cases of lung cancer were resected in our institute. Four cases of invasion into the aorta and seven cases of invasion into the superior vena cava were also operated and each vessels were reconstructed. Two pneumonectomies and nine lobectomies were performed. Four cases of squamous cell carcinoma, three of adenocarcinoma, three of large cell carcinoma and one of adenosquamous carcinoma were found on pathological examination. Four cases of N0, four of N2 and two of N3 were also detected on pathological lymph node evaluation. One case was stage IV with metastasis of ipsilateral axillar lymph nodes and the others were stage IIIB. With respect to pathological curability, absolutely noncurative operation was performed in all cases involving the aorta and absolutely noncurative in four and relatively curative three in cases involving the superior vena cava. During resection and reconstruction of the aorta, three cases received temporary blood supplying bypass and one case was underwent temporary clamping. Two cases developed bilateral lower extremity paralysis. During resection and reconstruction of the superior vena cava, one case underwent intraluminal temporary blood supplying bypass, two received sideclamping and four were reconstructed with double artificial grafts for bilateral innominate veins. Two cases who underwent resection of the aorta died within one year and one is alive after nine months of operation. Five cases who underwent resection of the superior vena cava died due to cancer. One case of N0 and relatively curative is alive in free of recurrence after seven years and six months of operation and another case who had brain metastasis resected is alive after two years and two months of pulmonary operation. Careful selection should be made for resection of the aorta because the prognosis is usually poor. In the resection of the superior vena cava, good prognosis is expected in some cases (N0, relatively curative) and quality of life is expected to improve with the prevention of the superior vena cava syndrome. Aggressive resection should be considered for lung cancer invasion into the superior vena cava.

Adult↗

PubMed1999

[Methicillin-resistant Staphylococcus aureus (MRSA) mediastinitis after cardiovascular operations].

We described three cases of poststernotomy mediastinitis caused by MRSA. The first case 74-year-old woman who had undergone emergent CABG was treated by continuous closed irrigation and consecutive omental transfer. She was suffered from multiple organ failure and died. The other two cases 50-year-old woman and 66-year-old man after double valves replacement and total aortic arch replacement were managed successfully with simultaneous combination of surgical debridement and placement of omental flap. It was seemed that early aggressive surgical procedures before deterioration of general conditions associated with improvement of mortality. Additionally, it was considered that to avoid residual dead space in presternum as well as in mediastinum was essential to decrease the morbidity and hospital stay, especially in case of MRSA infection.

Aged↗

PubMed1999

[Vitamin B12].

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Humans↗

PubMed1999

[Neopterin].

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Adolescent↗

PubMed1999

Diurnal height variations in growth of children: effects of gravity.

Many specialists in various scientific fields are interested in human growth and development of body sizes of children as a physiological phenomenon. However, our knowledge on human growth has not yet reached a solid foundation and consensus, and at present gravity potential is known to be a lasting condition that influences human lives in various ways. Therefore, more basic research is needed by examining detailed processes of human growth on the earth, with the effect of gravity in prospect. Although modern human growth study is constructed mainly on the basis of observations at one-year intervals, whether such long-interval observations can reveal a true statistical model of human growth is questionable. Togo and Togo (1982, 1989) observed growth in five siblings at one-month intervals for more than 15 years and found fluctuations observed from the start to the end of growth observations consisted of trend, regular, and irregular factors. This indicates the importance of examining human growth by short-interval observation. Thus, our study focused on diurnal variations of height, measuring twice daily, and discusses the effects of gravity on growth of children.

Adolescent↗

PubMed1998

Accelerated apoptosis of lymphocytes by augmented induction of Bax in SSI-1 (STAT-induced STAT inhibitor-1) deficient mice.

Growth, differentiation, and programmed cell death (apoptosis) are mainly controlled by cytokines. The Janus kinase-signal transducers and activators of transcription (JAK-STAT) signal pathway is an important component of cytokine signaling. We have previously shown that STAT3 induces a molecule designated as SSI-1, which inhibits STAT3 functions. To clarify the physiological roles of SSI-1 in vivo, we generated, here, mice lacking SSI-1. These SSI-1-/- mice displayed growth retardation and died within 3 weeks after birth. Lymphocytes in the thymus and spleen of the SSI-1-/- mice exhibited accelerated apoptosis with aging, and their number was 20-25% of that in SSI-1+/+ mice at 10 days of age. However, the differentiation of lymphocytes lacking SSI-1 appeared to be normal. Among various pro- and anti-apoptotic molecules examined, an up-regulation of Bax was found in lymphocytes of the spleen and thymus of SSI-1-/- mice. These findings suggest that SSI-1 prevents apoptosis by inhibiting the expression of Bax.

Animals↗

PubMed1998

Caspase-independent cell killing by Fas-associated protein with death domain.

The binding of Fas ligand to Fas recruits caspase 8 to Fas via an adaptor, FADD/MORT1, and activates a caspase cascade leading to apoptosis. Here, we describe a human Jurkat-derived cell line (JB-6) that is deficient in caspase 8. This cell line was resistant to the apoptosis triggered by Fas engagement. However, the multimerization of Fas-associated protein with death domain, through the use of a dimerizing system, killed the JB-6 cells. This killing process was not accompanied by the activation of caspases or DNA fragmentation. The dying cells showed neither condensation nor fragmentation of cells and nuclei, but the cells and nuclei swelled in a manner similar to that seen in necrosis. These results suggested that Fas-associated protein with death domain can kill the cells via two pathways, one mediated by caspases and another that does not involve them.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Interaction of Doc2 with tctex-1, a light chain of cytoplasmic dynein. Implication in dynein-dependent vesicle transport.

Doc2 has one Munc13-interacting domain at the N-terminal region and two C2-like domains interacting with Ca2+ and phospholipid at the C-terminal region. Doc2 consists of two isoforms, Doc2alpha and -beta. Doc2alpha is specifically expressed in neuronal cells and implicated in Ca2+-dependent neurotransmitter release, whereas Doc2beta is ubiquitously expressed and its function is unknown. We show here that both Doc2alpha and -beta interact with rat tctex-1, a light chain of cytoplasmic dynein, in both cell-free and intact cell systems. Overexpression of the N-terminal fragment of Doc2 containing the tctex-1-interacting domain induces changes in the intracellular localization of cation-independent mannose 6-phosphate receptor and its ligand, cathepsin D, which are transported from trans-Golgi network to late endosomes. Overexpression of the C-terminal fragment containing two C2-like domains shows the similar effect, but to a lesser extent, whereas overexpression of full-length Doc2 or the C-terminal fragment of rabphilin3 containing two C2-like domains does not show this effect. Because dynein is a minus-end-directed microtubule-based motor protein, these results suggest that Doc2, especially Doc2beta, plays a role in dynein-dependent intracellular vesicle transport.

Amino Acid Sequence↗

PubMed1998

Overexpression of cation-dependent mannose 6-phosphate receptor prevents cell death induced by serum deprivation in PC12 cells.

PC12 cells express well cation-independent mannose 6-phosphate receptors (CI-MPR), but not cation-dependent (CD)-MPR as much. To examine CD-MPR dependency of transport of cathepsins B and D to lysosomes in PC12 cells, we prepared the cells overexpressing CD-MPR. Immunoreactivity for cathepsin B became more distinct and larger in size in the transfected cells than in wild-type cells. No difference in the distribution of cathepsin D was seen between these two cells. The viability of the cells following serum deprivation was significantly higher in the transfected cells than in wild-type cells. This increased viability of the transfected cells was blocked by CA074, a specific inhibitor of cathepsin B, while pepstatin A suppressed the action of CA074. The results suggest that CD-MPR preferentially transport cathepsin B in PC12 cells, and cathepsins B and D participate in the regulation of PC12 cell apoptosis.

Amino Acid Sequence↗

PubMed1998

Participation of cathepsins B and D in apoptosis of PC12 cells following serum deprivation.

Cathepsin D, a lysosomal aspartic proteinase, has been shown to induce apoptosis of HeLa cells when overexpressed. To further understand regulatory mechanisms of cathepsin D-induced cell death, we examined whether lysosomal cysteine and aspartic proteinases are involved in apoptosis of PC12 cells following serum deprivation. In serum deprived culture, PC12 cells overexpressing cathepsin D died more rapidly than wild-type cells. When the active forms of cathepsins B and D were examined during the apoptotic process of wild-type cells, the amount of cathepsin B was drastically reduced 24 hr after the onset of culture, whereas that of cathepsin D considerably increased. The viability of PC12 cells overexpressing cathepsin B was significantly higher in serum-deprived culture than wild-type cells. In this situation, the amount of the cathepsin B protein did not decrease. The results suggest that there exists an apoptotic pathway regulated by lysosomal cathepsins B and D.

Animals↗

PubMed1998

Detection of activated Caspase-3 by a cleavage site-directed antiserum during naturally occurring DRG neurons apoptosis.

We prepared a cleavage site-directed antiserum against Caspase-3 (anti-p20/17), which reacts with the p20/17 fragment (p20/17) activated by cleavage but not proCaspase-3 (p32), and examined the relationship between the activation of Caspase-3 and apoptosis. We identified p20/17-positive cells where cell death occurs naturally: interdigits of the forelimbs, small intestine epithelium, thymus, trigeminal ganglia, and dorsal root ganglia of mouse embryos. Withdrawal of nerve growth factor induced the appearance of p20/17-positive cells with DNA fragmentation in the culture of dorsal root ganglia neurons, while DNA fragmentation was detected in both p20/17-positive and -negative neurons in dorsal root ganglia of mouse embryos. These results suggest that not only activation of Caspase-3 but also other molecular mechanism play a role in the naturally occurring dorsal root ganglia apoptosis. Cleavage site-directed antisera against Caspases will be useful for the analysis of the molecular mechanism of naturally occurring apoptosis during development.

Animals↗

PubMed1998

Purification and characterization of the active-site-mutated recombinant human mu-calpain expressed in baculovirus-infected insect cells.

Recombinant human mu-calpain whose active site Cys-115 was substituted with Ser was expressed in insect cells using baculovirus system. The mutant mu-calpain, purified using an affinity-column of calpastatin oligopeptides, had no proteolytic activities of autolysis and caseinolysis. The large subunit of the mutant mu-calpain was processed from the 80 kDa form to the 76 kDa form by the wild type calpain, supporting the intermolecular cleavage mechanism of procalpain during activation. Fluorescence polarization analysis revealed that the mutant mu-calpain retained high affinity toward fluorescein-labeled calpastatin domain 1. Fragmentation of the full-length calpastatin by the wild type calpain was enhanced by pre-incubating the inhibitor with the mutant calpain. The recombinant mutant calpain was suggested to retain the integrity of the high ordered structure of the wild type calpain.

Animals↗

PubMed1998

Degeneration in vivo of rat hippocampal neurons by wild-type Alzheimer amyloid precursor protein overexpressed by adenovirus-mediated gene transfer.

In an attempt to elucidate the pathological implications of intracellular accumulation of the amyloid precursor protein (APP) in postmitotic neurons in vivo, we transferred APP695 cDNA into rat hippocampal neurons by using a replication-defective adenovirus vector. We first improved the efficiency of adenovirus-mediated gene transfer into neurons in vivo by using hypertonic mannitol. When a beta-galactosidase-expressing recombinant adenovirus suspended in 1 M mannitol was injected into a dorsal hippocampal region, a number of neurons in remote areas were positively stained, presumably owing to increased retrograde transport of the virus. When an APP695-expressing adenovirus was injected into the same site, part of the infected neurons in the hippocampal formation underwent severe degeneration in a few days, whereas astrocytes near the injection site showed no apparent degeneration. These degenerating neurons accumulated different epitopes of APP, and beta/A4 protein (Abeta)-immunoreactive materials were undetected in the extracellular space. A small number of degenerating neurons showed nuclear DNA fragmentation. Electron microscopic examinations demonstrated that degenerating neurons had shrunken perikarya along with synaptic abnormalities. Microglial cells/macrophages were often found in close proximity to degenerating neurons, and in some cases they phagocytosed these neurons. These results suggest that intracellular accumulation of wild-type APP695 causes a specific type of neuronal degeneration in vivo in the absence of extracellular Abeta deposition.

Adenoviridae↗

PubMed1998

Prevention of hypoxic liver cell necrosis by in vivo human bcl-2 gene transfection.

Prevention of hypoxic cell death is a key to successful liver transplantation. We developed a new method for preventing liver hypoxic cell death by introducing an anti-cell death gene directly into rat livers. When the human bcl-2 gene (hbcl-2) was directly transfected into rat livers together with non-histone chromosomal protein high mobility group 1 (HMG1) by the hemagglutinating virus of Japan (Sendai virus; HVJ)-liposome method, human Bcl-2 protein (hBcl-2) was efficiently expressed. Electron microscopy and fluorescence microscopy revealed that hepatocytes expressing exogenous hBcl-2 were almost completely protected the hypoxic cell necrosis. The expression of the hBcl-2 also inhibited activation of caspase-3 (-like) proteases and liver dysfunction. Thus, we conclude that transfection of the hbcl-2 gene through HVJ-liposome method is useful to prevent liver cell necrosis induced by hypoxia. This finding could lead to new strategies to avoid the hypoxic cell death, the major problem in liver transplantation.

Animals↗

PubMed1998

A novel strategy for introducing exogenous bcl-2 into neuronal cells: the Cre/loxP system-mediated activation of bcl-2 for preventing programmed cell death using recombinant adenoviruses.

We have established a novel strategy for introducing exogenous Bcl-2 into neuronal cells that is mediated by Cre/loxP recombination using recombinant adenoviral vectors. An on/off-switching cassette for Bcl-2 (CALNLbcl-2) was designed to express Bcl-2 by recombinase Cre-mediated excisional deletion of a spacer DNA flanked by a pair of loxP sites. Exogenous Bcl-2 was clearly induced in PC12 cell lines carrying CALNLbcl-2 after infection with recombinant adenovirus producing recombinase Cre (AxCANCre). Dual infection with both AxCANCre and a recombinant adenovirus bearing CALNLbcl-2 showed efficient delivery of exogenous Bcl-2 into a hybrid motoneuronal cell line and primary chicken spinal motoneurons. The delivery of foreign Bcl-2 promoted survival of motoneurons in medium either containing or lacking trophic support. Thus, this strategy for delivery of exogenous Bcl-2 will be useful for studying neuronal death as well as for introducing foreign genes into postmitotic neurons under the control of recombinase Cre.

Adenoviridae↗

PubMed1998

Modulation of bone mass, strength, and turnover by a new benzamide compound, DU-6712, in adjuvant-induced arthritic rats.

To assess the reductions in bone mass, strength, and turnover in adjuvant-induced arthritic rats and to examine the effect of a new benzamide compound, DU-6712, on these parameters, a 28-day dosing experiment was performed using 8-week-old female Lewis rats. Arthritis was induced by injecting the adjuvant into the hind paw. The age-dependent increases in the body weight, lumbar bone mineral content and density (BMC and BMD) and compressive strength were disturbed in the arthritic rats. At 14 days, the histomorphometric parameters of bone formation (BFR/BS and BFR/BV) and the serum osteocalcin levels were significantly reduced compared with the baseline controls. However, the BMC values corrected for body weight did not differ significantly between the arthritic and normal rats, and the bone minerals were not reduced compared with the baseline controls. At 28 days, the parameters of bone minerals and strength of the lumbar body in the arthritic rats, both with and without correction for body weight, were significantly reduced compared with the baseline controls. The trabecular mineralizing surface remained significantly reduced and the osteoclast numbers were increased. DU-6712 at the doses of 5, 15, and 45 mg/kg, orally administered daily from the start of the experiment, significantly prevented the development of the chronic paw edema at 28 days. The reductions in the parameters of bone minerals, strength, and trabecular bone formation, and the increase in osteoclast number were alleviated by this agent. Age-dependent increases in the lumbar height, disturbed by the adjuvant injection, were also maintained. These data indicated that a 28-day period is necessary to obtain sufficient reductions in the bone mass and strength of the lumbar body concerning the model of secondary osteoporosis in adjuvant arthritic rats. DU-6712 was able to prevent these reductions by modulating the bone turnover in this arthritis model.

Animals↗