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Biomedical subjects

N Araki

Publications and source records attributed to N Araki.

At least 127 records · Page 7Linked to original sources
PubMed1993

Rapid and sensitive method of quantitation of bone gla protein mRNA using competitive polymerase chain reaction.

A method for sensitive quantitation of bone gla protein (BGP, osteocalcin) mRNA has been developed using competitive polymerase chain reaction after reverse transcription (competitive RT-PCR). The complementary DNA (cDNA) were transcribed from sample RNA was co-amplified in a PCR with a known amount of mutant BGP cDNA (competitor) using the identical oligonucleotide primers. The mutant cDNA with its unique restriction site allowed quantitation of sample and mutant PCR products after densitometric analysis of ethidium bromide-stained agarose gels. A linear relationship between initial sample BGP amount and the ratio of BGP to mutant BGP band intensity was obtained and used to make a standard curve to determine the initial BGP mRNA of unknown samples. These standard curves were made with known amounts of recombinant BGP cDNA. The competitive RT-PCR for BGP allows measurement of twofold differences in 1 and 2 micrograms total RNA and requires at least 10 times less sample RNA than usual Northern blotting. Moreover, heteroduplexes with one BGP strand and one mutant BGP strand formed as a result of high PCR cycles were quantifiable. This provided the advantages of rapid quantitation from ethidium bromide-stained gels without blotting, hybridization, or autoradiography. Multiple samples could be assayed for greater confidence in the results. The sensitivity, accuracy, and ease of the assay will facilitate analysis of BGP mRNA from a small amount of sample. The assay has been used to confirm the BGP mRNA changes with hormonal treatment in cultured cells and the age-related changes in whole tibia in vivo.

Animals↗
PubMed1993

Nematolysosomes (elongate lysosomes) in rat hepatocytes: their distribution, microtubule dependence, and role in endocytic transport pathway.

The distribution of lysosomes in rat hepatocytes was examined by three-dimensional electron microscopy combined with acid phosphatase (ACPase) cytochemistry. In the 2-microns-thick sections observed under 200- or 1000-kV TEM, it was apparent that ACPase activity localized on elongate lysosomes (we refer to them as nematolysosomes) with a diameter of 70-100 nm and lengths of several micrometers, as well as spherical lysosomes and trans-Golgi cisternae. Though most spherical lysosomes were located within the pericanalicular region, nematolysosomes were widely distributed throughout the hepatocytes. Typically, it appeared that the nematolysosomes elongated from the subsinusoidal region to the pericanalicular-Golgi complex area and they frequently formed a network at the cell periphery along the sinusoidal front. Furthermore, the formation of nematolysosomes was independent of new protein synthesis, but highly dependent on the integrity of microtubules. After a 6-8 h colchicine treatment, nematolysosomes were shrunk and/or fragmented, becoming roughly spherical lysosomes scattered throughout the cells. Nematolysosomes recovered their normal profiles after 24 h due to the reversible effect of the drug on microtubules. When the hepatocytes were exposed to horseradish peroxidase (HRP) in vitro or in vivo, HRP was quickly sequestered in nematolysosome-like structures via pinocytosis from the sinusoidal surface and transported to an area near the Golgi complex. These findings raise the possibility that the nematolysosomes engage in microtubule-dependent transport of macromolecules from the sinusoidal circulation to the Golgi complex area.

Acid Phosphatase↗
PubMed1993

The shape and distribution of lysosomes and endocytosis in the ciliary epithelial cells of rats.

The shape and distribution of lysosomes in the ciliary epithelium of rat eyes were examined by electron microscopy combined with acid phosphatase (ACPase) cytochemistry and three-dimensional observation of 2 microns-thick sections. ACPase activity was cytochemically localized in lysosomes and trans Golgi cisternae in the non-pigmented epithelial (NPE) and pigmented epithelial (PE) cells. In NPE cells, it was shown three-dimensionally, that most lysosomes had an elongate form, up to 5 microns in length, and a diameter of 70-100 nm. These elongate lysosomes (nematolysosomes) were predominantly located in the basal region of the cells. In contrast, PE cells had spherical lysosomes distributed at random throughout the cytoplasm. However, no nematolysosomes were seen in the PE cells. When the isolated ciliary processes were incubated in a medium containing horseradish peroxidase (HRP), HRP was incorporated into the nematolysosome-like structures by pinocytosis from the basal surface of the NPE cells. These findings suggest that nematolysosomes are associated with the pinocytotic activity of NPE cells. The pinocytosis-nematolysosomal route may be involved in the uptake and degradation of macromolecules from the aqueous humor in the posterior chamber.

Acid Phosphatase↗
PubMed1993

Relaxant effects of brain natriuretic peptide on guinea-pig tracheal smooth muscle.

1. The relaxant effects of brain natriuretic peptide (BNP) were investigated on guinea-pig tracheal smooth muscle. 2. Various amounts of BNP (10(-9)-10(-6) mol/L) showed concentration-dependent relaxant effects on resting tone, leukotriene D4 (LTD4; 10(-8) mol/L) and endothelin-1 (ET-1; 10(-8) mol/L) induced contraction of tracheal smooth muscle with EC50 values of 3.1 +/- 0.7 x 10(-8), 3.9 +/- 1.0 x 10(-8) and 3.5 +/- 1.0 x 10(-8) mol/L, respectively. 3. BNP increased tissue cyclic GMP levels in tracheal smooth muscle concentration dependently (187 +/- 26 fmol/mg protein in control, 334 +/- 77 fmol/mg protein at 10(-8) mol/L, 680 +/- 54 fmol/mg protein at 10(-7) mol/L, 2162 +/- 133 fmol/mg protein at 10(-6) mol/L). 4. With the addition of BNP, tissue cyclic GMP levels reached a maximum at 1-3 min. The relaxation of tracheal smooth muscle began at 1 min and reached a maximum level at 5 min after the superfusion of BNP (10(-6) mol/L). The elevation of cyclic GMP preceded the relaxation of tracheal smooth muscle. 5. These results suggest that BNP may have a potent relaxant effect on tracheal smooth muscle and this effect may be mediated by cyclic GMP level.

Animals↗
PubMed1993

beta 2-Microglobulin modified with advanced glycation end products is a major component of hemodialysis-associated amyloidosis.

beta 2-Microglobulin (beta 2M) is a major constituent of amyloid fibrils in hemodialysis-associated amyloidosis, a complication of long-term hemodialysis patients. Amyloid fibril proteins were isolated from connective tissues forming carpal tunnels in hemodialysis patients with carpal tunnel syndrome. Two-dimensional polyacrylamide gel electrophoresis and Western blotting demonstrated that most of the beta 2M forming amyloid fibrils exhibited a more acidic pI value than normal beta 2M. This acidic beta 2M was also found in a small fraction of beta 2M in sera and urine from these patients, whereas heterogeneity was not observed in healthy individuals. We purified acidic and normal beta 2M from the urine of long-term hemodialysis patients and compared their physicochemical and immunochemical properties. Acidic beta 2M, but not normal beta 2M, was brown in color and fluoresced, both of which are characteristics of advanced glycation end products (AGEs) of the Maillard reaction. Immunochemical studies showed that acidic beta 2M reacted with anti-AGE antibody and also with an antibody against an Amadori product, an early product of the Maillard reaction, but normal beta 2M did not react with either antibody. Incubating normal beta 2M with glucose in vitro resulted in a shift to a more acidic pI, generation of fluorescence, and immunoreactivity to the anti-AGE antibody. The beta 2M forming amyloid fibrils also reacted with anti-AGE antibody. These data provided evidence that AGE-modified beta 2M is a dominant constituent of the amyloid deposits in hemodialysis-associated amyloidosis.

Amyloidosis↗
PubMed1993

Experimental implant-related osteomyelitis treated by antibiotic-calcium hydroxyapatite ceramic composites.

The efficacy of locally implanted antibiotic-calcium hydroxyapatite ceramic composites was investigated for the treatment of experimentally produced, implant-related osteomyelitis in rats. High concentrations of antibiotics were detected at the site of infection and bacteria were eradicated without removal of the metal implants. Parenteral antibiotics and surgical debridement, alone or in combination with antibiotic-impregnated acrylic bone cement, all failed to eradicate the infections.

Animals↗
PubMed1993

[A case of drug induced pneumonitis caused by oral etoposide].

We report a case of drug induced pneumonitis caused by oral etoposide. A 63-year-old man was admitted to our hospital in August 1991 because of low grade fever and dyspnea. He underwent right upper lobectomy on Nov. 27th, 1990 for lung cancer (squamous cell carcinoma), and courses of adjuvant chemotherapy (CBDCA, IFX, etoposide) during the following admission period. He was discharged on Feb. 14th, 1991, and as an outpatient, oral etoposide (25 mg/day) was administered for about 7 months (6,125 mg in total). Chest X-ray film on admission showed reticulonodular shadows in bilateral lung fields, and computed tomography showed diffuse interstitial shadows. Blood gas analysis showed marked hypoxemia (PaO2 breathing room air was 48.4 Torr). Transbronchial lung biopsy revealed edema of the alveolar walls and marked proliferation of type II alveolar epithelial cells, suggesting cytotoxic reaction. After termination of etoposide administration and following steroid pulse therapy, both clinical symptoms and hypoxemia were ameliorated. To our knowledge, this is the first report of etoposide-induced pneumonitis.

Administration, Oral↗
PubMed1993

An improved technique for observing both membranous organelles and cytoskeleton in saponin-extracted cells.

Detergent-extraction of soluble cytoplasmic proteins under low osmotic pressure induces severe morphological damage, particularly on membranous organelles, although this processing is often required to observe cytoskeletal elements in the cells. In the present study, the osmotic adjustment of the rinsing solution and fixative, which were used after the detergent-extraction using saponin, has greatly improved the ultrastructure of membranous organelles. This improved method allowed us to observe both cytoskeletal elements and the fine structure of membranous organelles by electron microscopy.

Animals↗
PubMed1992

Discovery of bone gamma-carboxyglutamic acid protein in mineralized scales. The abundance and structure of Lepomis macrochirus bone gamma-carboxyglutamic acid protein.

The mineralized scale of the freshwater sunfish Lepomis macrochirus (bluegill) contains a Gla protein. The protein was identified in extracts of scale by a new colorimetric assay for Gla-containing proteins. The protein was purified by gel filtration chromatography followed by reversed phase high performance liquid chromatography (HPLC). Several tests establish the identity of scale Gla protein and bone Gla protein (BGP). First, the proteins exhibit identical mobilities on electrophoresis and by reversed phase HPLC. Second, they have identical amino-terminal amino acid sequences. Finally, identical peptides are generated by proteolytic digestion. The 45-residue amino acid sequence of the bone Gla protein from L. macrochirus has a high sequence homology with swordfish, as well as homology to mammalian bone Gla protein. The BGP of bluegill shares with swordfish BGP a truncated NH2 terminus and an extended COOH terminus. These features may be unique to fish, as they have not been observed in terrestrial vertebrates. The bluegill BGP is the first vitamin K-dependent protein to contain a non-gamma-carboxylated residue to the NH2-terminal side of all of its Gla residues. In all other vitamin K-dependent proteins, Gla always appears to the NH2-terminal side of the first Glu. The implications of this result are discussed. The bluegill rib bone is curiously enriched in BGP, as are other mineralized tissues of this species. One hypothesis is that this may be due to the acellular nature of the bone in this species. The abundance of BGP in the bones of this fish may provide clues to the unknown function of this bone protein.

Amino Acid Sequence↗
PubMed1992

Relaxant effect of pituitary adenylate cyclase-activating polypeptide on guinea-pig tracheal smooth muscle.

We investigated the relaxant effect of the pituitary adenylate cyclase-activating polypeptide with 27 residues (PACAP27) and with 38 residues (PACAP38) on guinea-pig tracheal smooth muscle. Both forms of PACAP showed dose-dependent relaxant effects. The EC50 of PACAP27 was 8.7 +/- 1.9 x 10(-8) M and that of PACAP38 was 6.8 +/- 1.0 x 10(-8) M. Both increased cyclic AMP levels dose dependently and the elevation of cyclic AMP preceded the relaxation of tracheal smooth muscle. There was a marked difference in the duration of action of the two peptides. PACAP38 showed a longer-lasting relaxation compared to PACAP27. Furthermore PACAP38 maintained significantly higher levels of cyclic AMP, with cyclic AMP levels at 60 min after a 5-min exposure to PACAPs (10(-6) M) being 14.0 +/- 1.4 pM/mg protein for PACAP27 and 35.9 +/- 2.4 pM/mg protein for PACAP38. These results suggest that PACAP27 and PACAP38 may be novel potent relaxants in tracheal smooth muscle and their relaxant effect might be mediated by cyclic AMP. However PACAP38 had a longer-lasting action on relaxation of tracheal smooth muscle and production of tissue cyclic AMP than PACAP27.

Animals↗
PubMed1992

Immunochemical evidence for the presence of advanced glycation end products in human lens proteins and its positive correlation with aging.

Prolonged incubation of protein with reducing sugar proceeds through a series of reactions involving early stage products to the advanced glycation end products with fluorescence, brown color, and cross-linking. Known collectively as the Maillard reaction, these changes have been suggested as factors in diabetic complications and the aging process. The early stage products have been demonstrated in vivo, but evidence for the presence in vivo of the advanced glycation end products has been limited. We sought to provide immunochemical evidence by the preparation and use of polyclonal and monoclonal antibodies to these end products (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332) as probes to identify and quantitate such compounds in human lens crystallins. Neither of the antibodies reacted with extracts from infant lenses, but fractions from adult lenses showed a significant reactivity, correlating with lens age. Our findings provide the first immunochemical evidence that human lens crystallins contain advanced glycation end products and that these products increase with tissue age.

Adult↗
PubMed1992

Slow release of anticancer drugs from porous calcium hydroxyapatite ceramic.

We have developed a new delivery system for sustained release of an anticancer drug (cis-platinum) by enclosure into blocks of porous calcium hydroxyapatite ceramic. The slow release of this drug from this system was confirmed in in vitro experiments. When this system was implanted into normal back muscle, or the tibia, sustained release of cis-platinum was observed during a 12-week period after implantation. The diffusion rate of cis-platinum into blood and other organs (liver, kidney, brain) was less than 10% of that at the implanted site. This delivery system placed into experimental tumors of mice also showed a uniform release of anticancer drug for more than 3 months. Inhibition of tumor growth was more marked after local implantation of this system than after intraperitoneal administration of cis-platinum. These results indicate that this new approach to a drug delivery system may well have an important role in cancer chemotherapy. In bone tumors it is attractive because the mechanical strength of calcium hydroxyapatite ceramic permits partial surgical excision and replacement of the bone defect at the same time.

Animals↗
PubMed1992

Effect of superoxide dismutase on intracellular calcium in stroke.

To clarify the relationship between calcium metabolism and free radical damage during the reperfusion period following ischemia, we investigated the effect of superoxide dismutase (SOD) on changes in cytosolic free calcium, cortical blood flow, and histologic changes following focal cerebral ischemia and reperfusion in 12 cats. Using indo-1, a fluorescent intracellular Ca2+ indicator, we simultaneously measured changes in the Ca2+ signal ratio (400:500 nm), NADH signal (464 nm), and reflectance (340 nm) during ultraviolet excitation (340 nm) directly from the cortex in vivo. The middle cerebral artery (MCA) was occluded for 1 h; only cats in which the EEG amplitude was depressed to less than 10% of control during the occlusion were entered into the study. Starting 2 min prior to release of the occlusion and continuing for 4 min, SOD (10,000 U/kg) was slowly infused in six cats, while in six cats, the vehicle only was infused. During MCA occlusion, the Ca2+ signal ratio increased significantly in both groups with no significant difference between the groups. During reperfusion, the Ca2+ signal ratio remained at a high level in the vehicle-treated group, while in the SOD-treated group, the Ca2+ signal ratio decreased. There was a statistically significant difference between the two groups at 10, 20, and 30 min after reperfusion (p less than 0.01). The histologically damaged area in the SOD-treated group was significantly smaller than that in the vehicle-treated group (p less than 0.01). These data suggest that the histoprotective action of SOD may be due to its ability to attenuate increases in intracellular calcium during the recirculation period following focal cerebral ischemia.

Animals↗
PubMed1992

The effect of hyperglycemia on intracellular calcium in stroke.

The effect of hyperglycemia on cytosolic free calcium ([Ca2+]i) during temporary focal cerebral ischemia was investigated in cats using a fluorometric technique. The middle cerebral artery (MCA) was occluded for a period of 1 h, after which the clip was removed. In seven animals, plasma glucose was raised to 500-700 mg/dl by infusion of a 50% glucose solution starting 30 min after MCA occlusion, while eight animals were kept normoglycemic during and following occlusion. MCA occlusion induced a significant, but identical, elevation of the [Ca2+]i signal ratio (400/506 nm) in both the normoglycemic group (from 1.40 to 1.97 +/- 0.34, p less than 0.01) and in the hyperglycemic group (from 1.40 to 2.00 +/- 0.53, p less than 0.01) at the end of the occlusion. Between 10 and 30 min after reopening, the [Ca2+]i signal ratio decreased to control levels in the normoglycemic group (1.40 +/- 0.11 and 1.36 +/- 0.08 at 10 and 30 min after reopening, respectively), but remained elevated in the hyperglycemic group (1.69 +/- 0.18 and 1.65 +/- 0.21 at 10 and 30 min after reopening, respectively). There was a statistically significant difference between the two groups (p less than 0.01). These data suggest that hyperglycemia may be harmful to calcium recovery during the early recirculation period following focal cerebral ischemia.

Animals↗
PubMed1992

Calcium hydroxyapatite ceramic used as a delivery system for antibiotics.

Porous blocks of calcium hydroxyapatite ceramic were evaluated as delivery systems for the sustained release of antibiotics. We tested gentamicin sulphate, cefoperazone sodium, and flomoxef sodium in powder form placed in a cylindrical cavity in calcium hydroxyapatite blocks, using in vitro studies of elution and in vivo studies in rats. Gentamicin sulphate gave a maximum concentration within the first week, which gradually decreased but was still effective at 12 weeks, when 70% of the antibiotic had been released. Even at this stage the antibiotic concentration from a 75 mg dose was five times the minimum inhibitory concentration for staphylococci. In the in vivo studies the release of gentamicin sulphate into the normal bone of rats was at similar rates and levels. The bacteriocidal activity of the drugs was not affected by packing into calcium hydroxyapatite ceramic and the blocks were completely biocompatible on histology. This new system overcomes the disadvantages of other drug delivery systems, avoiding thermal damage to the antibiotics and a second operation for the removal of the carrier. Some mechanical strength is provided by the ceramic and healing may be accelerated by bone ingrowth into its micropores.

Animals↗
PubMed1992

A case of amylase-producing lung cancer.

A case of lung cancer with hyperamylasemia was studied. Small cell carcinoma was diagnosed histologically. The salivary gland and pancreas had no clinical involvement in the hyperamylasemia. Saliva-type amylase was dominant as observed from amylase isozyme patterns in the serum and tumorous tissue. Levels of amylase were higher in tumorous tissue than in normal lung tissue. Immunohistochemical study showed amylase localized in tumor cells. Observation of the ultrastructure revealed electron-dense granules in the cytoplasm of the tumor cells. Findings suggested that amylase was being produced by the lung cancer in this case.

Amylases↗