[Structures and biological activities of heparin-like molecules].
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Amino Acid Sequence↗
Biomedical subjects
M Okayama
Publications and source records attributed to M Okayama.
Purification and characterization of novel heparan sulfate proteoglycans produced by murine erythroleukemia cells in the growing phase.
Murine erythroleukemia cells (Friend erythroleukemia cells of a C-10-6 line) synthesized sulfated glycosaminoglycans consisting mainly of heparan sulfate (more than 95%) with a small amount of chondroitin 4-sulfate. The heparan sulfate occurred as proteoglycans, of which the cell-associated component was separated into urea-insoluble (UI) and urea-soluble (US) fractions. The UI proteoglycan consisted of a single homogeneous molecular species with an estimated Mr of 360,000 (C(UI)PG), whereas the US component was composed of two subfractions: a homogeneous species with an Mr of 280,000 (C(US)PGI) and a mixture of compounds with Mr values of less than 80,000 (C(US)PGII), which were isolated in yields of about 110, 340, and 80 micrograms of hexuronate (HexUA), respectively, from 1.37 g of an acetone powder prepared from 5.7 x 10(9) cells in the logarithmic phase of growth. The proteoglycan released into the medium (12 liters) was a single homogeneous species with an Mr of 320,000 (MPG) which was purified in a yield of 500 micrograms of hexuronate. The major, cell-associated proteoglycan, C(US)PGI, had very high contents of serine and glycine, accounting for approximately 80% of the total amino acids. This proteoglycan as well as the other two large proteoglycans, C(UI)PG and MPG, were highly resistant to degradation by various proteinases. These three proteoglycans, C(UI)PG, C(US)PGI, and MPG, had heparan sulfates with estimated Mr values of 32,000, 27,000, and 30,000. On the other hand, the Mr of the smaller proteoglycan, C(UI)PGII, was not significantly different before and after beta-elimination, indicating that it contains only a small peptide, if any. The heparan sulfate of this proteoglycan consisted of smaller and heterogeneous molecular species with Mr values of 26,000, 20,000, and 4,000. Digestion of these heparan sulfates with heparitinase I plus II resulted in almost complete depolymerization and gave six unsaturated disaccharides, delta HexUA-GlcNAc, delta HexUA-Glc-NAc(6-SO4), delta HexUA-GlcNSO3, delta HexUA-GlcNSO3 (6-SO4), delta HexUA(2-SO4)-GlcNSO3, and delta HexUA(2-SO4)-GlcNSO3(6-SO4). The relative amounts of these disaccharides generated from the individual heparan sulfates showed that an average ratio of sulfate residues to repeating disaccharide units of the C(US)PGII-derived heparan sulfate (0.97) was significantly higher than those of the other three large proteoglycan-derived glycosaminoglycans (0.54-0.70).
Amino Acids↗
Close association between tumour cells and vascular basement membrane in gastric cancers with liver metastasis. An immunohistochemical and electron microscopic study with special attention to extracellular matrices.
To investigate morphological features valuable in estimating the propensity of gastric cancer to metastasize to the liver, we examined the primary tumours from 49 surgically resected advanced gastric cancers (24 with liver metastasis) and 45 autopsy cases, 19 with liver metastasis. We paid special attention to extracellular matrices--connective tissue stroma and basement membrane (BM)--using immunohistochemistry and electron microscopy. Type IV collagen staining showed that in differentiated carcinomas neoplastic glands were occasionally located in close proximity to the BM of thin-walled tumour blood vessels in back-to-back fashion. In poorly differentiated lesions, tumour cells were also oriented toward the vascular BM in pseudorosette-like pattern. Type III collagen staining and electron microscopy showed that in such regions tumour cells, with continuous or discontinuous BM, were immediately adjacent to vascular BM with no connective tissue stroma in between. On occasion tumour cells were in direct contact with vascular BM. These close associations were often found in carcinomas with a medullary growth pattern, irrespective of the degree of histological differentiation. However, they were virtually never seen in their benign counterpart. Of the resected cases, all 24 with liver metastasis showed this association, whereas only 10 of 25 (40%) without liver metastasis did so (P less than 0.001). In the autopsied cases, a similar positive correlation was observed between liver metastasis and this association. Furthermore, the tumor cells showing this juxtaposition showed evidence of vascular invasion. These results suggest that the close association between tumour cells and vascular BM is specific to the malignant neoplasm, and may be related to liver metastasis. Immunohistochemistry can be a great help in estimating the probability of liver metastasis.
Basement Membrane↗
Substance P and neurokinin A in human nasal mucosa.
The tachykinins substance P (SP) and neurokinin A (NKA) were studied in human inferior turbinate nasal mucosa by radioimmunoassay, immunohistochemistry, and autoradiography and for their effect upon mucus release in an in vitro culture system in order to infer their potential functions in the upper respiratory tract. Similar amounts of SP (1.03 +/- 0.12 pmol/g wet weight; mean +/- SEM; n = 26) and NKA (0.76 +/- 0.23; n = 7) were found. NKA and SP immunoreactive nerve fibers were found in the walls of arterioles, venules, and sinusoids and as individual fibers in gland acini, near the basement membrane, and in the epithelium. [125I]SP bound to arterioles, venules, and glands. [125I]NKA bound only to arterioles. In short-term explant culture of fragments of human nasal mucosa, both 1 microM SP and 1 microM NKA stimulated release of [3H]glucosamine-labeled respiratory glycoconjugates. These results indicate that SP and NKA have similar distributions in nociceptive sensory nerves in human nasal mucosa. The distribution of [125I]SP binding sites is consistent with a role for SP as a vasodilator and mucous secretagogue. The presence of [125I] NKA binding sites on vessels suggests a primary role for NKA in regulating vasomotor tone.
Amino Acid Sequence↗
Treatment of status asthmaticus with intravenous magnesium sulfate.
The bronchodilating effect of magnesium sulfate (MgSO4) was studied in two patients with status asthmaticus, who were intubated and mechanically ventilated by a respirator. Airway resistance was continuously monitored by the respiration-controlled interruption technique. After administration of 0.5 mmol/min MgSO4 intravenously, airway resistance decreased from 17 to 9, and from 13 to 8 mmHg/L/s in the two patients, respectively, and piping rales diminished or disappeared. We conclude that while corticosteroid therapy requires several hours to demonstrate significant effects in status asthmaticus, MgSO4 is of great benefit in the rapid improvement of airflow obstruction.
Adolescent↗
Isolation and characterization of proteoglycans synthesized by cultured mesangial cells.
Rat mesangial cells selected by long-term culture of glomeruli exhibited a hill and valley appearance in the confluent state and were stained with antibodies against vimentin and desmin, suggesting that they are smooth muscle-like mesangial cells. The glycoconjugates produced by the cells were metabolically labeled with [35S]sulfate and [3H]glucosamine and extracted with 4 M guanidine HCl containing 0.5% Triton X-100. The radiolabeled glycoconjugates were separated on DEAE-Sephacel and compared with those synthesized by glomeruli labeled in the same conditions. Of the three major sulfated glycoconjugates, sulfated glycoprotein (17% of the total 35S-labeled macromolecules), heparan sulfate proteoglycan (35%), and chondroitin sulfate proteoglycan (30%) synthesized by glomeruli, the cultured mesangial cells synthesized mainly chondroitin sulfate proteoglycan (more than 90%). After purification by CsCl density-gradient centrifugation, the chondroitin sulfate proteoglycan from the cell layer was separated on Bio-Gel A-5m into three molecular species with estimated Mr values of 230,000, 150,000, and 40,000-10,000, whereas that released into the medium consisted of a single species with an Mr of 135,000. In the beta-elimination reaction, the former two larger proteoglycans released chondroitin sulfate chains with Mr of an apparent 30,000 and the latter from the medium released the glycosaminoglycan chains with an Mr of 36,000. The Mr of the smallest proteoglycan from the cell layer was not significantly changed after beta-elimination, indicating that this species had only a small peptide, if any. Analysis with chondroitinase AC-II and ABC demonstrated that all the chondroitin sulfates were copolymers consisting of glucuronosyl-N-acetylgalactosamine (65-74%) having sulfate groups at position 4 (53-57%) or positions 4 and 6 (10-14%) of hexosamine moieties and iduronosyl-N-acetylgalactosamine (21-26%) having sulfate groups at position 4 (17-23%) or positions 4 and 6 (about 3%) of hexosamine moieties; namely chondroitin sulfate H type. These characteristics of the chondroitin sulfate H proteoglycans synthesized by the cultured mesangial cells were very similar to those of the proteoglycans synthesized by glomeruli. Thus, we conclude that most, if not all, of the glomerular chondroitin sulfate proteoglycans are synthesized by mesangial cells. The cultured mesangial cells were also found to synthesize hyaluronic acid at a similar level to chondroitin sulfate proteoglycan. Based on the characteristics of this glycosaminoglycan, we discuss the possible role of hyaluronic acid produced by mesangial cells.
Animals↗
Vasoactive intestinal peptide in human nasal mucosa.
Vasoactive intestinal peptide (VIP), which is present with acetylcholine in parasympathetic nerve fibers, may have important regulatory functions in mucous membranes. The potential roles for VIP in human nasal mucosa were studied using an integrated approach. The VIP content of human nasal mucosa was determined to be 2.84 +/- 0.47 pmol/g wet weight (n = 8) by RIA. VIP-immunoreactive nerve fibers were found to be most concentrated in submucosal glands adjacent to serous and mucous cells. 125I-VIP binding sites were located on submucosal glands, epithelial cells, and arterioles. In short-term explant culture, VIP stimulated lactoferrin release from serous cells but did not stimulate [3H]glucosamine-labeled respiratory glycoconjugate secretion. Methacholine was more potent than VIP, and methacholine stimulated both lactoferrin and respiratory glycoconjugate release. The addition of VIP plus methacholine to explants resulted in additive increases in lactoferrin release. Based upon the autoradiographic distribution of 125I-VIP binding sites and the effects on explants, VIP derived from parasympathetic nerve fibers may function in the regulation of serous cell secretion in human nasal mucosa. VIP may also participate in the regulation of vasomotor tone.
Cells, Cultured↗
Effect of duration of vagal stimulation on shortening velocity of in vivo canine trachealis muscle.
We studied shortening velocity of in vivo canine trachealis muscle contracted by bilateral vagus nerve stimulation, as a function of duration of contraction. The cervical trachea was transected at two locations, cut at the ventral portion, and opened. One side of the cartilage cut was connected to a force transducer and the other to a lever with a given weight as an afterload, the length of which was measured by a linear displacement transducer. Bilateral vagosympathetic trunks were stimulated by supramaximal electrical impulses. With vagal stimulation, the trachealis muscle started to contract isometrically and at a given time the muscle was allowed to contract isotonically by unlocking a stopper at a given afterload. The shortening velocity was reduced with longer duration of active state. Ten sec after vagal stimulation the maximum force was 730 +/- 105 g/cm2 (mean +/- S.D.) and maximum velocity at zero load calculated by Hill's equation was 0.092 l0/sec. We conclude that the force-velocity relationship of in vivo canine trachealis muscle stimulated by vagus nerves exhibits a time-dependency similar to that in vitro.
Animals↗
Breathing 30 per cent oxygen attenuates bronchial responsiveness to methacholine in asthmatic patients.
The effect of breathing 30% oxygen on bronchial responsiveness to inhaled methacholine was studied in 30 asthmatic patients using a direct graphical recording method. An aerosol of methacholine solution was inhaled during tidal breathing by measuring respiratory resistance (Rrs) continuously, and dose-response curves were obtained on two separate days during air and 30% oxygen inhalation. Oxygen inhalations for 20 min caused no change in baseline Rrs. The threshold dose of methacholine, the dose which increased Rrs from the baseline value while breathing 30% oxygen, increased significantly compared with that while breathing air (p less than 0.01). No significant difference was found between the mean values of the slopes of the dose-response curves of the two groups. End-expiratory oxygen concentration (FETO2) during air breathing decreased significantly from 13.7% to 12.0% at the point where methacholine dosage reached threshold. This study suggests that hyperoxia attenuates the bronchial responsiveness to methacholine in asthmatic patients.
Adolescent↗
Sulfated glycoproteins synthesized by the corneal epithelium of chick embryo having the same molecular weights as cytokeratin polypeptides.
The previous study from this laboratory demonstrated that the corneal epithelium of 19-d-old chick embryo synthesizes two classes of sulfated glycoconjugates consisting of sulfated glycoproteins and proteoglycans (Yonekura, H., Oguri, K., Nakazawa, K., Shimizu, S., Nakanishi, Y., & Okayama, M. (1982) J. Biol. Chem. 257, 11166-11175). The present study demonstrated that when the sulfated glycoproteins labeled metabolically with [35S]sulfate and [3H]glucosamine were analyzed by SDS-PAGE, the 70,000 component (accounting for approximately 30% of the 35S and 35% of the 3H of the total sulfated glycoprotein) co-migrated with five major proteins with apparent molecular weights (Mrs) of 70,000, 66,000, 58,000, 51,000, and 48,000, which together accounted for about 57% of the total tissue protein. All five proteins cross-reacted with an antibody against human sole keratin, indicating that they are cytokeratin polypeptides of the corneal epithelium. Amino acid analysis demonstrated that they had high contents of glycine, serine, glutamic acid, leucine, and aspartic acid. Two-dimensional tryptic peptide maps indicated that they were all different. Analysis of radiolabeled materials released by alkaline borohydride treatment of the sulfated glycoproteins which were synthesized in the presence and absence of tunicamycin and co-purified with the five cytokeratin polypeptides, revealed that they contained both N- and O-glycosidically linked sulfated oligosaccharides. All the results obtained in the present study indicate that the five sulfated glycoproteins are similar, if not identical, to the cytokeratin polypeptides. This is consistent with the result in the accompanying paper that these sulfated glycoproteins are localized intracellularly.
Animals↗
The localization of sulfated glycoconjugates synthesized by the corneal epithelium of chick embryos.
The localization of sulfated glycoconjugates in the corneal epithelium of 19-d-old chick embryo was investigated biochemically using epithelia labeled in vitro with [35S]sulfate, which exhibited autoradiographically a similar distribution of silver grains to that labeled in ovo. The radiolabeled tissues were dissociated into single cells by incubation in 0.25% trypsin containing 0.02% EDTA at 37 degrees C for 40 min. The proteoglycans and sulfated glycoproteins which were associated with the cells and those released into the dissociation medium were separated by DEAE-Sepharose CL-6B and analyzed on Sepharose CL-6B and SDS-PAGE. About 86% of the proteoglycans was released into the dissociation medium and more than 50% of the cell-associated ones was affected by trypsin. This indicates that the proteoglycans are mostly localized in an extracellular compartment. On the other hand, the extent of release of sulfated glycoproteins into the medium on dissociation of tissues was distinctly different depending upon their molecular weight (Mr): almost all of the sulfated glycoproteins of the family with Mr 48,000-70,000 (32% of the total sulfated glycoproteins) were recovered as intact molecules with the cells, whereas approximately 50% of those with Mr 70,000-150,000 (36%) and about 70% of those with Mr over 150,000 (28%) were released into the dissociation medium. These results indicate that the family with Mr 48,000-70,000 is localized intracellularly and that with Mr 70,000-150,000 in a compartment poorly affected by trypsin; in contrast to those, that with Mr more than 150,000 is localized in an extracellular compartment like the proteoglycans.
Animals↗
Isolation and characterization of chondroitin 6-sulfate proteoglycans present in the extracellular matrix of rabbit bone marrow.
The authors' previous studies showed that the glycosaminoglycans present in rabbit bone marrow were composed of chondroitin 6-sulfate (79%) and hyaluronic acid (16%). Immunohistochemically the chondroitin 6-sulfate was demonstrated to be in bone marrow matrix constructing hematopoietic microenvironment. In this study the authors isolated and characterized these glycosaminoglycans in their macromolecular form (i.e., proteoglycans). Bone marrow of 3-month-old rabbits was defatted with organic solvents containing proteinase inhibitors at -20 degrees C, and proteoglycans were extracted from the defatted tissue with 4 mol/L guanidine HCl containing the proteinase inhibitors. After extensive dialysis of the extract against 7 mol/L urea, more than 90% of hexuronate-containing materials was recovered in the urea-soluble fraction. The proteoglycans were purified from the urea-soluble fraction by diethyl aminoethyl (DEAE)-Sephacel chromatography, CsCl density-gradient centrifugation, and Bio-Gel A-5m gel filtration, then were rechromatographed on DEAE-Sephacel and on Bio-Gel A-5m. The proteoglycans were separated into three molecular species with different mol wts that were assessed to be 46,000, 16,000, and 8,300 by sedimentation-equilibrium centrifugation. Amino acid analyses of these proteoglycans revealed that serine and glycine accounted for approximately 60% of the total amino acids common to the three proteoglycans. The glycosaminoglycan side chains of these proteoglycans were converted stoichiometrically into unsaturated 6-sulfated disaccharide by digestion with chondroitinase AC-II, indicating that they were fully sulfated chondroitin 6-sulfate. Their apparent mol wts were estimated by gel filtration on Bio-Gel A-0.5m to be 10,900, 14,400, and 7,700. Computation of these results, taken together with their biochemical composition, revealed that the largest proteoglycan, PG-II, consisted of four chains of the chondroitin 6-sulfate and the core peptide with mol wt of approximately 4,000. The smaller two proteoglycan subpopulations, PG-I and PG-III, consisted of a single glycosaminoglycan chain linked to a small peptide with mol wts of approximately 500 and 1,000, respectively.
Amino Acids↗
Bronchodilating effect of intravenous magnesium sulfate in bronchial asthma.
The bronchodilating effect of magnesium sulfate (MgSO4) was studied in ten asthmatic patients with mild attacks. In five patients, 0.5 mmol/min of MgSO4 was administered intravenously for 20 minutes, and the time courses of respiratory resistance, forced vital capacity, and forced expiratory volume at 1 s were studied. In another five patients, MgSO4 dose-response curves were obtained. Soon after administration began, MgSO4 relieved bronchoconstriction in a dose-dependent manner. Maximum responses (mean +/- SE) of respiratory resistance, forced vital capacity, and forced expiratory volume were 71% +/- 3%, 117% +/- 5%, and 118% +/- 1% of initial values, respectively, and were similar to the effects of additional albuterol inhalation. The infusion of MgSO4 also improved dyspnea and piping rales in three other asthmatic patients with a severe attack. We conclude that intravenous infusion of MgSO4 produces a rapid and marked bronchodilation in both mild and severe asthma and may be a unique bronchodilating agent.
Adult↗
A new method of inhalation challenge with propranolol: comparison with methacholine-induced bronchoconstriction and role of vagal nerve activity.
To establish a safe procedure for examining propranolol-induced bronchoconstriction, we have developed a new method for performing inhalation challenge with propranolol. Monitoring respiratory resistance during tidal breathing with continuous inhalation of propranolol in 1.5-fold increasing concentrations from 0.78 to 30 mg/ml for 1 minute at each concentration, we tested 43 subjects with stable asthma and 10 normal subjects. We also compared bronchial responsiveness with responsiveness to inhaled methacholine on separate days. In addition, to determine the role of vagal nerve activity in propranolol-induced bronchoconstriction, we studied the effect of atropine. Inhaled propranolol caused dose-related bronchoconstriction in all subjects with asthma but not in normal subjects. None of the subjects suffered severe asthmatic attack during the test, which was performed in 15 minutes or less. The minimum cumulative dose of methacholine and of propranolol, at the point where respiratory conductance began to decrease, was not significantly correlated. Increased respiratory resistance was reversed by atropine in 70% of the subjects with asthma with marked individual differences. These data suggest that, although in most subjects with asthma, vagal nerve activity contributes in varying degree to bronchoconstriction, other constricting factors may contribute in the remaining subjects. It is also suggested that the mechanism of bronchial response to propranolol differs from that of the nonspecific airway reactivity estimated by methacholine challenge.
Adolescent↗
Chondroitin 6-sulfate proteoglycans constructing hemopoietic microenvironment in bone marrow.
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Animals↗
Isolation, characterization, and localization of glycosaminoglycans in rabbit bone marrow.
The glycosaminoglycans that exist in rabbit bone marrow were analyzed chemically, and their in situ localization was studied immunohistochemically. Femoral bone marrow of 3-month-old rabbits was defatted with organic solvents. Glycosaminoglycans were prepared from the defatted tissue after its digestion with pronase, treatment with mild alkali, and then digestion with DNase-I. The tissue contained glycosaminoglycans equivalent to 195 mg of hexosamine per femur, which accounted for 27.3% of the total hexosamine in the tissue. Studies with hyaluronidase from Streptomyces hyalurolyticus and chondroitinase ABC showed that the glycosaminoglycans were composed of hyaluronic acid (16% of the total glycosaminoglycan) and chondroitin 6-sulfate (79%). The chondroitin 6-sulfate was separated on Bio-Gel A-0.5m gel into two molecular species with mol wt of greater than 12,000 (Kd greater than 0.2) and approximately 8,000 (Kd = 0.47). Bone marrow digested with chondroitinase ABC and then treated with three monoclonal antibodies 4/8/9-A-2, 5/6/3-B-3, and 5/6/1-B-5, which were specific for unsaturated 4-sulfated, 6-sulfated, and nonsulfated disaccharide structures, respectively, at the nonreducing end of chondroitin sulfate chains, reacted with only 5/6/3-B-3. This result indicated that the chondroitin sulfate, isomer in the bone marrow is chondroitin 6-sulfate, consistent with the biochemical results. The chondroitin 6-sulfate was localized mainly in the extracellular compartment and was considered to be involved in construction of the hemopoietic microenvironment in the bone marrow.
Animals↗
Purification and characterization of human platelet proteoglycan.
Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.
Amino Acids↗
Effect of sublingual isosorbide dinitrate on sputum volume and viscoelasticity in chronic obstructive lung disease.
The effect of isosorbide dinitrate (ISDN) on sputum volume and sputum viscoelasticity in 12 patients with chronic obstructive lung diseases (COLD) was investigated by the double-blind randomized cross-over method with matched placebo. Each patient was given either sublingual ISDN 5 mg or placebo at 9 a.m. and sputum was collected for 3 h before and after 9 a.m. The dynamic viscoelasticity of the sputum was measured using a coaxial cylinder rheometer at an angular frequency of 0.1 rad/s. ISDN significantly increased sputum volume, and it caused a significant decrease in dynamic viscosity and in dynamic elasticity compared to placebo. The findings suggest that ISDN might be useful as an expectorant in the treatment of COLD when abundant and viscous sputum is present.
Adult↗