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Biomedical subjects

S Inoue

Publications and source records attributed to S Inoue.

At least 1,099 records · Page 61Linked to original sources
PubMed1989

Structure, composition, and assembly of basement membrane.

Basement membranes are thin layers of matrix separating parenchymal cells from connective tissue. Their ultrastructure consists of a three-dimensional network of irregular, fuzzy strands referred to as "cords"; the cord thickness averages 3-4 nm. Immunostaining reveals that the cords are composed of at least five substances: collagen IV, laminin, heparan sulfate proteoglycan, entactin, and fibronectin. Collagen IV has been identified as a filament of variable thickness persisting after the other components have been removed by plasmin digestion or salt extraction. Heparan sulfate proteoglycan appears as sets of two parallel lines, referred to as "double tracks," which run at the surface of the cords. Laminin is detected in the cords as diffuse material within which thin wavy lines may be distinguished. The entactin and fibronectin present within the cords have not been identified as visible structures. The ability of laminin, heparan sulfate proteoglycan, fibronectin, and entactin to bind to collagen IV has been demonstrated by visualization with rotary shadowing and/or biochemical studies. Incubation of three of these substances-collagen IV, laminin (with small entactin contamination), and proteoglycan-at 35 degrees C for 1 hr resulted in a precipitate that was sectioned for electron microscopic examination and processed for gold immunolabeling for each of the three incubated substances. Three structures are present in the precipitate: 1) a lacework, exclusively composed of heparan sulfate proteoglycan in the form of two parallel lines, similar to double tracks; 2) semi-solid, irregular accumulations, composed of the three initial substances distributed on a cord network; and 3) convoluted sheets, which are also composed of the three initial substances distributed on a cord network but which, in addition, have the uniform appearance and thickness of the lamina densa of basement membrane. Hence these sheets are closely similar to the main component of authentic basement membranes.

Animals↗
PubMed1989

Association of fibronectin with the microfibrils of connective tissue.

The association of fibronectin with the microfibrils of connective tissue was examined in the zonular fibers of the mouse eye by immunohistochemical methods at the light and electron microscopic level. Mouse eyes fixed in formaldehyde were embedded either in paraffin for immunostaining by the peroxidase-antiperoxidase (PAP) method or in Lowicryl for immunolabeling by antirabbit globulin antibodies bound to 5 or 15 nm gold particles. Ultrastructural studies were also carried out after glutaraldehyde perfusion. Both the PAP and immunogold procedures demonstrated the association of fibronectin with microfibrils. After immunolabeling with 5 nm gold particles, examination at high magnification localized fibronectin to fine filaments that appeared to be attached to the surface of microfibrils. The filaments extended outward singly or formed loose aggregates. Their diameter ranged from 1.2 to 3 nm, with a mean of 1.5 nm. Because of their similarity to the fibronectin molecules previously described after rotary shadowing, the filaments were likely to be fibronectin molecules themselves. Since fibronectin is known to have high affinity for the amyloid P component, a model is presented in which fibronectin filaments are bound to the amyloid P component making up the tubular core of microfibrils in mice. Evidence is presented that fibronectin filaments may link microfibrils to one another and thus insure the continuity and strength of zonular fibers. More generally, it is likely that connective-tissue microfibrils, whether or not inserted into elastic fibers, are bonded through fibronectin to surrounding cells, collagen fibrils, or proteoglycans, and thus insure cohesion among connective tissue elements.

Actin Cytoskeleton↗
PubMed1989

The intersegmental anastomoses between spinal nerve roots.

Frequency and morphological characteristics of the intersegmental anastomoses between the spinal roots were investigated in 100 adult spinal cords from the cervical to lumbar segments. The frequencies of dorsal root anastomoses were 61, 7, and 22% of the cervical, thoracic, and lumbar cords, respectively. On the other hand, the incidence of anastomosis between the ventral roots was significantly low in both the cervical (10%) and lumbar (5%) cords as compared with that between the dorsal roots in these regions (P less than .01). No anastomotic connections were found in the thoracic ventral roots. The anastomoses were classified into four types: types A and B; the borders between the spinal segments were cranially and caudally shifted, respectively; type C, anastomosis attached to the spinal cord at the border between the segments; and type D, anastomosis bridged between the adjacent rootlets and coursing parallel to the cord. The proportions of the four types at each spinal level disclosed that type A was most commonly observed compared with the other three types in the dorsal roots (P less than .01). In the lumbar cord, the proportion of type C anastomosis between the dorsal roots increases noticeably toward the sacral level, while a complementary decrease occurred in type A. In the cervical ventral roots, types C and D predominated over the other two types (P less than .01). The lumbar ventral roots contained types B and/or C. Type D anastomosis occurred exclusively in the cervical segments.

Humans↗
PubMed1989

Improved separation of alpha chains of collagen type I, type III, and type V by noninterrupted electrophoresis using thioglycolic acid as a negatively charged reducer.

Improved separation of alpha chains of collagen type I (alpha 1 [I]2 alpha 2[I]), type III(alpha 1[III]3), and type V (alpha 1[V]alpha 3[V])was achieved by noninterrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a negatively charged reducer, thioglycolic acid. The thioglycolic acid, added to the running buffer of the cathodic reservoir, in the middle of electrophoresis quickly migrated in the gel anode, reducing interchain disulfide linkages in collagen type III and dissociating it into its alpha chain monomer, alpha 1[III], without an interruption of electrophoresis. The alpha chain, alpha 1[III], migrated more slowly than the alpha 1 [I] and alpha 2[I] chains of collagen type I, resulting in an excellent separation of alpha 1[III] from alpha 1[I]. The mobility of alpha 1[III] could be controlled by varying the time of thioglycolic acid addition to the running buffer. This enabled us not only to separate alpha 1[III] from alpha 1[I] and alpha 1[V], but also to precisely quantitate these alpha chains, even at low protein loading of mixed samples.

Collagen↗
PubMed1989

Abnormal organization of keratin intermediate filaments in cultured keratinocytes of epidermolysis bullosa simplex.

Distinctive abnormality in the organization of keratin intermediate filaments (KIFs) was found for the first time in cultured epidermal keratinocytes from two patients with hereditary epidermolysis bullosa simplex (EBS), which showed cleavages above the basement membrane zone due to the fragility of basal cells. KIFs in EBS keratinocytes revealed an irregular radial arrangement composed of sparse but thick KIF bundles. Furthermore, these KIF bundles in many cells changed into numerous ball-like keratin aggregates and disappeared beyond these keratin aggregates in the peripheral cytoplasm. Electron microscopy of cultured EBS keratinocytes showed that many ball-like structures consisting of fine filaments or granules or homogeneous substances were scattered in the peripheral regions of the cell attaching to the dish, and intermediate filaments appeared to be emanating from or surrounding the structures. These ball-like keratin aggregates have never been observed in normal human keratinocytes.

Adult↗
PubMed1989

Long-term prognosis of surgical patients with hepatocellular carcinoma.

We reviewed 139 resected patients with hepatocellular carcinoma at our clinic between 1963 and 1987, and using the 118 cases for the period between 1963 and 1986, we analyzed the prognostic factors that influenced the long-term prognosis by comparing the survival curves. Significant differences in the survival patterns were noted when analysed on the basis of the preoperative indocyanine green maximal removal rate (greater than 0.4 mg kg-1 min-1 versus less than 0.4 mg kg-1 min-1), tumor size (greater than 5 cm versus less than 5 cm, etc.) and the existence of tumor capsule. The recurrence of carcinoma was the main cause of death of 32 patients (56%), who died after being discharged from hospital. To improve the prognosis of patients with surgically treated hepatocellular carcinoma, postoperative multidisciplinary treatment is mandatory.

Carcinoma, Hepatocellular↗
PubMed1989

Human breast carcinoma (ZR-75-1) serially transplanted into nude mice--with reference to estradiol dependency and sensitivity to tamoxifen.

Tumor cells of the ZR-75-1 line (1 X 10(7) in 0.5 ml) were inoculated into the right thigh muscles of BALB/c female nude mice which were, at the same time given 1.0 mg of estradiol subcutaneously. After the transferable strain had been established, the tumors were then transplanted into female and male nude mice either with or without estradiol treatment. Although no exponential tumor growth was observed in the untreated male and female nude mice, no complete rejection was found during the experiments. The estrogen receptor of this strain was positive and the growth of ZR-75-1 was dependent on exogenous estradiol. ZR-75-1 in the nude mouse was insensitive to tamoxifen treatment, given as 5 mg/kg intramuscularly twice a week, suggesting that dependency on estradiol is not necessarily correlated with endocrine sensitivity to tamoxifen.

Animals↗
PubMed1989

Isolation and characterization of a novel type of sialoglycoproteins (hyosophorin) from the eggs of medaka, Oryzias latipes: nonapeptide with a large N-linked glycan chain as a tandem repeat unit.

We found a novel type of sialoglycoprotein (SGP) with apparent molecular mass ranging from 15,000 to 100,000 Da in the unfertilized eggs of the medaka fish, Oryzias latipes. From fertilized eggs we isolated the corresponding sialoglycopeptides of apparent molecular weight 7000. The amino acid and carbohydrate compositions of these glycoproteins and glycopeptides are very similar, if not identical, and they contain 90%, by weight, of carbohydrate, the predominant sugars being Gal, GlcNAc, and NeuAc. The chemical and physical data indicate that 15- to 100-kDa SGPs are made up of tandem repeat structures whose repeating unit is 7-kDa sialoglycopeptide, and, upon fertilization, higher molecular weight SGPs undergo proteolytic depolymerization to the least structural unit, 7-kDa sialoglycopeptide. As is the case with polysialoglycoproteins (PSGP) found in salmonid fish eggs, a novel family of sialoglycoproteins has been proven to be a major component of cortical alveoli of medaka eggs, namely, hyosophorin. However, we found that they differ markedly from PSGPs (salmonid fish egg hyosophorins) in terms of the carbohydrate composition. The chemical composition and the results of Smith degradation indicate that SGP contains one large N-linked glycan chain per repeat unit. We have determined the amino acid sequence of 7-kDa sialoglycopeptide: Asp-Ala-Ala-Ser-Asn*-Gln-Thr-Val-Ser, where * indicates the asparagine residue to which a large glycan chain consisting of Fuc2Man3Gal15GlcNac9NeuAc6 is attached. The direct experimental evidence for the presence of a polyprotein structure suggests that the covalent nature of the higher molecular weight SGPs should be expressed as [Asp-Ala-Ala-Ser-Asn*-Gln-Thr-Val-Ser]N, where N = 2 to 14 but for the major fraction N = 12.

Amino Acid Sequence↗
PubMed1989

Involvement of adrenergic mechanism in hyperglycemia due to SCN stimulation.

Previously we found that in rats, electrical stimulation of the suprachiasmatic nucleus (SCN) of the hypothalamus elicited hyperglycemia associated with hyperglucagonemia without immediate hyperinsulinemia. To clarify the mechanism of these responses, we examined the effects of blockers of the autonomic nervous system on these responses. Hexamethonium, a ganglion blocker, suppressed the hyperglycemic and hyperglucagonemic responses to electrical stimulation of the SCN. Both bunazosin, an alpha 1-adrenergic blocker, and yohimbin, an alpha 2-adrenergic blocker, increased the level of insulin before stimulation, but only the latter suppressed the hyperglycemic and hyperglucagonemic responses. Propranolol, a beta-adrenergic blocker, partially inhibited the responses. These findings suggest that alpha 2- and beta-adrenergic mechanisms are involved in the hyperglycemic and hyperglucagonemic responses to SCN stimulation.

Adrenergic alpha-Antagonists↗
PubMed1989

The incubation of laminin, collagen IV, and heparan sulfate proteoglycan at 35 degrees C yields basement membrane-like structures.

Three basement membrane components, laminin, collagen IV, and heparan sulfate proteoglycan, were mixed and incubated at 35 degrees C for 1 h, during which a precipitate formed. Centrifugation yielded a pellet which was fixed in either potassium permanganate for ultrastructural studies, or in formaldehyde for Lowicryl embedding and immunolabeling with protein A-gold or anti-rabbit immunoglobulin-gold. Three types of structures were observed and called types A, B, and C. Type B consisted of 30-50-nm-wide strips that were dispersed or associated into a honeycomb-like pattern, but showed no similarity with basement membranes. Immunolabeling revealed that type B strips only contained heparan sulfate proteoglycan. The structure was attributed to self-assembly of this proteoglycan. Type A consisted of irregular strands of material that usually accumulated into semisolid groups. Like basement membrane, the strands contained laminin, collagen IV, and heparan sulfate proteoglycan, and, at high magnification, they appeared as a three-dimensional network of cord-like elements whose thickness averaged approximately 3 nm. But, unlike the neatly layered basement membranes, the type A strands were arranged in a random, disorderly manner. Type C structures were convoluted sheets composed of a uniform, dense, central layer which exhibited a few extensions on both surfaces and was similar in appearance and thickness to the lamina densa of basement membranes. Immunolabeling showed that laminin, collagen IV, and proteoglycan were colocalized in the type C sheets. At high magnification, the sheets appeared as a three-dimensional network of cords averaging approximately 3 nm. Hence, the organization, composition, and ultrastructure of type C sheets made them similar to the lamina densa of authentic basement membranes.

Basement Membrane↗
PubMed1989

Effects of physiological oxygen environment on drug-induced cell lethality of multicellular tumor spheroids from human lung cancer.

Advanced malignant tumors of certain histological types contain a hypoxic and necrotic core. Multicellular tumor spheroids (MTS) have the characteristics of chronically hypoxic cells in the center. We studied the effects of physiological oxygen environment on MTS growth and the cell lethality produced by doxorubicin (DXR) and cisplatin (DDP). MTS were made from 2 human lung cancer cell lines; PC-6 small cell and PC-10 squamous cell carcinoma, and grown for 2, 3 or 4 weeks; either in 5% CO2/air or 5% 02/5% CO2/90% N2. They were exposed to graded concentrations of DXR for 1 hr and cell lethality was determined by clonogenic assay. In the physiological oxygen environment MTS growth was retarded for both cell lines. PC-6 MTS grown in physiological oxygen environment were more sensitive to DXR than those developed in air. The differential sensitivity was most pronounced with the 2 week old MTS and gradually narrowed with increasing MTS size. In contrast, PC-10 MTS developed in the physiological oxygen environment were more resistant to DXR than those in air; the differences were again most pronounced in 2 week old MTS. There were little differences in cell kill effects of DDP, irrespective of cells being in monolayer or in MTS and growing in air or in physiological oxygen environment. These observations are consistent with the interpretation that cells in PC-6 MTS are scarcely affected by the physiological oxygen environment but easily affected by DXR, whereas cells in PC-10 MTS responded vice versa.

Antineoplastic Agents↗
PubMed1989

Site-specific DNA damage induced by nickel(II) ion in the presence of hydrogen peroxide.

DNA damage induced by Ni(II) plus H2O2 was investigated by a DNA sequencing technique using 32P-5'-end-labeled DNA fragments obtained from human c-Ha-ras-1 protooncogene. Ni(II) induced strong DNA cleavage in the presence of H2O2 even without piperidine treatment. Piperidine-labile sites were induced frequently at cytosine, thymine and guanine residues, and rarely at adenine residue. Diethylene-triamine N,N,N',N",N"-pentaacetic acid inhibited the DNA damage. In experiments with singlet oxygen scavengers, sodium azide and dGMP inhibited the DNA damage completely, whereas neither 1,4-diazabicyclo[2.2.2]octane nor dimethylfuran inhibited it. Among hydroxyl radical scavengers, dimethylsulfoxide and sodium formate inhibited the DNA damage considerably, whereas ethanol and mannitol did not. Methionine and methional inhibited the DNA damage completely. The results suggest that Ni(II) ion binds to DNA and subsequently reacts with H2O2 to form active species, which cause DNA damage. The possibility of Ni(II) plus H2O2-mediated DNA damage in vivo is discussed relative to the molecular mechanism of nickel carcinogenesis.

Base Sequence↗
PubMed1989

pH dependence of the reaction rate of His 48 with p-bromophenacyl bromide and of the binding constant to Ca2+ of the monomeric forms of intact and alpha-NH2 modified phospholipases A2 from Trimeresurus flavoviridis.

The phospholipase A2 of Trimeresurus flavoviridis was found to show monomer-dimer equilibria. Under conditions where the enzyme exists predominantly in the monomeric form, the chemical reaction rate of p-bromophenacyl bromide (BPB) with the catalytic group, His 48, was studied at 25 degrees C and ionic strength 0.2 by measuring the residual enzymic activity using a fluorescent substrate, 1,2-bis[4-(1-pyreno)butanoyl]-sn-glycero-3-phosphorylcholine (diPBPC). The pH-dependence curve of the reaction rate for the intact enzyme was practically the same as that for the modified enzyme, in which the N-terminal alpha-NH2 group had been selectively converted into an alpha-keto group. The pH-dependence curves were monophasic (sigmoidal) with a midpoint at pH 7.53, which corresponds to the pKa value of His 48. The pH dependences of the binding constants of Ca2+ to the intact and the alpha-NH2 modified enzymes were also studied at 25 degrees C and ionic strength 0.2 by measuring the changes in the tryptophyl fluorescence and/or aromatic CD spectra. The pH-dependence data for the modified enzyme were interpreted in terms of participation of Asp 49 (pKa 5.40) and His 48 (pKa 7.53), assuming that the protonation of Asp 49 competes with the Ca2+ binding. The pH-dependence data for the intact enzyme were similarly interpreted in terms of participation of the alpha-NH2 group (pKa 9.40) in addition to that of Asp 49 (pKa 5.40) and His 48 (pKa 7.53).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetophenones↗
PubMed1989

Interactions of monodispersed and micellar substrates with a phospholipase A2 from Trimeresurus flavoviridis.

Bindings of the phospholipase A2 from Trimeresurus flavoviridis to the monodispersed and micellar n-alkylphosphorylcholines (n-CnPC) were studied at 25 degrees C and ionic strength 0.2 by the aromatic CD and tryptophyl fluorescence methods, respectively. The bindings to micelles of the substrate analog were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 9-13. The binding constant to the micelle was about 40 times greater than it was to the monodispersed substrate. The binding constant to the micellar substrate analog increased on the binding of Ca2+ to the enzyme and decreased on modification of the N-terminal alpha-NH2 group, whereas the binding to the monodispersed substrate analog was independent of pH, of the Ca2+ binding, and of the chemical modification of the alpha-NH2 group. The kinetics of the hydrolyses of monodispersed and micellar dihexanoylphosphatidylcholines (diC6PC) were studied at 25 degrees C and ionic strength 0.2 by the pH-stat method in the presence of saturating amounts of Ca2+. The catalytic center activity, kappa cat, as well as the binding constant, 1/Km, for the micellar substrate, were found to be much greater than those for the monodispersed substrate. The binding constant, 1/Km, of the monodispersed substrate was independent of pH; this was in good agreement with that of the substrate analog described above. The pH-dependence curve of kappa cat for the monodispersed substrate exhibited two transitions, one below pH 6.5 and the other above pH 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Isolation and fundamental properties of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis.

Phospholipase A2 inhibitor was purified from the blood plasma of Habu, Trimeresurus flavoviridis, by Sephadex G-200 gel filtration, DEAE-cellulose chromatography, and Blue-Sepharose CL-6B column chromatography. The purified inhibitor was shown to be a glycoprotein with a molecular weight of about 100K. It was found to consist of four subunits whose molecular weights were around 20-24K. In order to examine the inhibition mechanism of the inhibitor, the interaction of the inhibitor with a phospholipase A2 from T. flavoviridis venom was examined by Sephadex G-100 gel filtration. One inhibitor molecule was found to bind directly to one phospholipase A2 molecule in both the presence and absence of Ca2+. The inhibitor inhibited the phospholipase A2 from T. flavoviridis venom with an apparent dissociation constant, Ki, of 1.7 X 10(-10) M, but not the porcine pancreas enzyme or the Agkistrodon halys blomhoffii enzyme belonging to the same family, Crotalidae, as T. flavoviridis, or the phospholipase C from Bacillus cereus.

Animals↗
PubMed1989

Long-term results of spinal instrumentation surgery for scoliosis five years or more after surgery, in patients over twenty-three years of age.

From 1967 to 1981, 311 scoliotic patients treated at Chiba University, with a minimum follow-up of 5 years and aged more than 23 years, were selected for this study. Seventy-two were males and 239 were females, whose age at operation ranged from 9 to 44 years, with an average of 16.5 years. The operative procedures were one-stage Harrington procedure in 179 cases, staged procedure in 108 cases, anterior procedure in 20 cases, and combined anteroposterior fusion in four cases. Including 198 patients examined physically, a total of 263 patients (54 males and 209 females) have responded to a questionnaire or were interviewed by telephone. In general, 85% of the patients were satisfied with the result of the operation for the control of spinal deformity, improvement of the cosmesis, and increase in physical and social activity.

Activities of Daily Living↗
PubMed1989

Correlation between cell-cell contact formation and activation of protein kinase C in a human squamous cell carcinoma cell line.

Formation of desmosomal cell-cell contact associated with reorganization of keratin intermediate filaments (KIFs) was observed when cultured cells of a cell line of human skin squamous cell carcinoma were transferred from low (0.07 mM) calcium to high (1.87 mM) calcium medium. At low calcium, cells were dispersed without desmosomal cell-cell contact and the KIFs were mostly concentrated around the nucleus. After 15 min of the transfer, cells contacted each other and formed small colonies and the KIFs initiated to show a radial arrangement. In addition to the cell-cell contact formation and rearrangement of KIFs, the transfer induced fourfold increase of particulate-associated protein kinase C (C-kinase) activity. When 12-O-tetradecanoyl phorbol-13-acetate (PMA), which specifically activates C-kinase, was added to the cells grown at low calcium medium, cell-cell contact formation and radial arrangement of KIF bundles almost identical to those induced by the transfer to high calcium medium were observed. These data suggest a correlation between an increase in C-kinase activity and formation of cell-cell contacts associated with rearrangements of KIFs.

Calcium↗