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A novel captodatively substituted benzoquinone methide, 7-cyano-7-(phenyl)benzoquinone methide (3), was synthesized successfully as an isolable crystal at room temperature. 3 polymerized spontaneously on dissolving it in highly polar, basic solvents such as DMSO and DMF. The acid-catalyzed 1,6-addition reaction of 3 was investigated. Acid-catalyzed 1,6-addition reactions of 3 with phenol and N,N-dimethylaniline afforded 1:1 adducts. In the reaction with hydrogen chloride, a 1,6-addition adduct was also formed. Homopolymerizations of 3 and 7,7-di(cyano)benzoquinone methide (1a) with radical, anionic, and cationic initiators were investigated. 3 and 1a are homopolymerizable with an anionic initiator but not with radical and cationic ones. 3 is copolymerizable spontaneously with styrene in an alternating fashion. The copolymer structure of 3 with styrene was determined on the basis of the spectral data of model compounds and the polymerization mechanism was discussed.

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.

Background: Histamine is metabolized mainly by histamine N-methyltransferase (HMT) to N^@t-methylhistamine in human nasal mucosa. Human HMT cDNA has been cloned and expressed in COS cells. The purpose of this study was to determine the localization of HMT mRNA in human nasal mucosa. Methods: The fragment (nucleotide residues 430-1055) of human HMT cDNA was subcloned in a Bluescript vector (Stratagene, La Jolla, Calif.), and HMT sense and anti-sense RNA probes were made with T"7 and T"3 RNA polymerases. In situ hybridization with digoxigenin-labeled RNA probes was performed on surgical specimens of human nasal turbinates. Results: HMT mRNA was localized in cells in the epithelium and submucosa, and densely in endothelial cells of vessels. No HMT mRNA was identified in the submucosal glands. The presence of HMT mRNA was confirmed by Northern blot analysis, and HMT activities were also detected in nasal mucosa. Conclusion: Our study indicates that endothelium expresses HMT mRNA, whereas cells in the epithelium and submucosa, which remain unidentified, are an additional source of HMT mRNA. (J ALLERGY CLIN IMMUNOL 1995;95:96-102.)

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.

The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, xβ-D-xylosides and β-D-galactosides to the incubation medium. β-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, β-D-xylosides and β-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.

Glomeruli isolated from rat kidney were incubated with [14C]glucosamine and [35S]sulfate. Linear incorporation of [14C]glucosamine into total glycosaminoglycans was observed during incubation up to 24 h. More than 95% of the 35S-labeled sulfated glycoconjugates were extracted from the tissue with 4 M guanidine HCl, 50 mM sodium acetate, pH 6.0, and 0.5% Triton X-100, and separated clearly on DEAE-Sephacel into three major fractions, i.e. sulfated glycoprotein (11% of the total radioactivity), proteoheparan sulfate (33%), and proteochondroitin sulfate (38%) fractions. The molecular weight of the 35S-labeled proteoheparan sulfate thus isolated was estimated to be about 185,000, whereas that released into the medium was estimated to be about 87,000. When the 35S-labeled heparan sulfate isolated on Sephadex G-75 after mild alkaline borohydride treatment was digested with a combination of heparitinase and heparinase, approximately 70% of the radioactivity was converted to 2-acetamido-2-deoxy-4-O-(alpha-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D- glucose.

The bronchodilating effect of 5 mg sublingual isosorbide dinitrate (ISDN) was studied in 10 patients with bronchial asthma, using the doubleblind randomised cross-over method with matched placebo. In a further 20 asthmatics the effect of sublingual ISDN was compared with that of metaproterenol given by a metered dose inhaler to a total dose of 2.25 mg, again using the cross-over method. The forced oscillation method was used to measure respiratory resistance (Rrs) and spirometry was used to measure vital capacity (VC) and forced expiratory volume in one second (FEV1). 5 minutes after administration of ISDN Rrs had decreased (p<0.05) and VC (p<0.01) and FEV1 (p<0.01) were significantly increased. The changes were still present after 15, 30 and 60 min. The placebo had no significant effect. ISDN increased FEV1 less than metaproterenol, and the difference between them was statistically significant (p<0.05). However, there was no significant difference between ISDN and metaproterenol in the improvement in Rrs and VC. Of the total of 30 patients, 11 experienced headache and 4 had transient hypotension after ISDN administration. These side effects subsided spontaneously. It was concluded that sublingual ISDN had a bronchodilating effect in stable asthmatics.

Incorporation of sulfate into sulfated proteoglycans by isolated chicken chondrocytes was inhibited up to 74% by transformation with the Rous sarcoma virus, and a similar inhibitory effect was observed on acetate incorporation into chondroitin sulfate. Slower sedimenting sulfated proteoglycans appear after the viral transformation. The ratio of chondroitin 4-sulfate to chondroitin 6-sulfate in these slower sedimenting sulfated proteoglycans was different from that of normal chondrocytes, but the chain lengths of sulfated glycosaminoglycans produced by normal chondrocytes and transformed chondrocytes were not significantly different. Chondrocytes were also infected with a temperature-sensitive mutant of RSV, ts LA24, which has a temperature-sensitive lesion in the transforming gene. Hyaluronic acid production by these cells was increased, and the slower sedimenting sulfated proteoglycan was produced only at the permissive temperature.

The sulfated proteoglycans synthesized by definitive chondroblasts in cultured 10-day chick vertebral or epiphyseal cartilages were characterized by their sedimentation profile in a sucrose gradient and their susceptibility to chondroitinase ABC (EC 4.2.2.4; chondroitin ABC lyase). These sulfated proteoglycans were indistinguishable from those synthesized by definitive chondroblasts that emerge from older cultures of somites plus notochord or in older cultures of limb buds. The sulfated proteoglycans of these definitive chondroblasts are readily distinguished from those synthesized by their mother cells, the presumptive chondroblasts, or those synthesized by dedifferentiated or bromodeoxyuridine-suppressed chondroblasts. However, the sulfated proteoglycans synthesized by presumptive chondroblasts or by dedifferentiated or bromodeoxyuridine-suppressed chondroblasts cannot be dintinguished by these techniques from those synthesized by (i) blastodisc cells, (ii) fibroblasts, (iii) spinal cord cells, or (iv) skeletal, cardiac, or smooth muscle cells. Addition of glycosaminoglycans or collagen to the medium did not induce somite or limb presumptive chondroblasts to synthesize the chondroblast-unique sulfated proteoglycans. Cells moving from the presumptive chondroblast compartment into the chondroblast compartment acquire not only the option to initiate the synthesis of chondroblast-unique collagen chains, but also the capacity to synthesize chondroblast-unique sulfated proteoglycans.

The corneal epithelia isolated from 19-day-old chick embryos were incubated with [35S]sulfate and [3H]glucosamine. More than 95% of the isotope-labeled sulfated glycoconjugates were extracted with 4 M guanidine/HCl, 50 mM sodium acetate, pH 6.0, and 0.5% Triton X-100 and separated on DEAE-Sepharose CL-6B into two major fractions, i.e. sulfated glycoprotein and sulfated proteoglycan fractions. Mild alkali treatment of the sulfated proteoglycan fraction and characterization of the sulfated glycosaminoglycans enabled the identification of heparan sulfate ([35S], 81%; [3H], 91%) and chondroitin sulfate ([35S], 19%; [3H], 9%). Fractionation of the sulfated glycoprotein fraction on Sepharose CL-6B resulted in the separation of four sulfated glycoproteins with apparent molecular weights of 70,000, 130,000, 280,000, and over 2,000,000. The products of alkaline borohydride treatment of the sulfated glycoproteins separated into three fractions on Sephadex G-75, two fractions contained sulfated oligosaccharides, and the third one contained inorganic sulfate and N-acetyl-galactosaminitol. Incorporation of [35S]sulfate into higher molecular weight fraction of the two fractions was selectively inhibited by tunicamycin, whereas that incorporated into the smaller molecular weight fraction was not inhibited by the drug and, rather, was activated. These results suggest that the former contains N-glycosidically linked sulfated glycopeptides, and the latter is derived from O-glycosidically linked sulfated oligosaccharide side chains.

This study addresses the characterization of heparan sulphates of the basement-membrane proteoglycans in tumour formed after the subcutaneous implantation of Lewis-lung-carcinoma-derived different metastatic clones (P29, LM12-3 and LM60-D6 clones with low, medium and high metastatic potentials respectively). Heparan sulphate proteoglycans (125-158 micrograms of hexuronate/g dry weight of tissue) were isolated from chondroitin ABC lyase digests of a proteoglycan fraction obtained after DEAE-Sephacel chromatography of tissue extracts. The proteoglycans were separated into three molecular species by Sepharose CL-4B chromatography followed by CsCl-density-gradient centrifugation: large proteoglycans with an estimated M(r) of 820,000-130,000, which consisted of two components with low (< 1.34 g/ml; PGII-M) and high (> 1.37 g/ml; PGII-B) density, and a small proteoglycan with an M(r) of less than 80,000 (PGIII). Of these, only the PGII-M proteoglycan (34-37 micrograms of hexuronate/g dry weight) reacted with the antiserum against proteoglycan of Engelbreth-Holm-Swarm-tumour basement membrane, and represented, therefore, a basement-membrane proteoglycan. Digestion with heparan sulphate lyases I and II of the heparan sulphates (M(r) 36,000) from the PGII-M proteoglycan of the three tumours resulted in almost complete depolymerization to give six unsaturated disaccharides identified as 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-D-glucose, 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-D-glucose and 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-6-O-sulpho-D-glucose. Comparison of the relative amounts of these disaccharides produced from the three tumour-derived heparan sulphates demonstrated that the degree of sulphation of the heparan sulphates correlated with the degree of morphological organization of the tumour basement membranes; the heparan sulphate from the more highly metastatic tumour with more highly organized basement membrane exhibited a higher degree of overall sulphation along the glycosaminoglycan chains, which was due to an increased content of the three repeating disaccharides having 6-O-sulphated glucosamine residues.

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.

To study the temporal aspect of visual processing of senile dementia of the Alzheimer type (SDAT), we recorded the steady-state visual evoked potentials (VEPs) to LED goggle stimulation at five different temporal frequencies in 14 patients with SDAT and 14 control subjects. For comparison, we also studied 14 patients with vascular dementia (VD). All subjects were women, and their ages were closely matched. Temporal frequencies were 5, 10, 15, 20, and 30 Hz. The steady-state VEPs were Fourier analyzed, and both the amplitude and phase of the first and second harmonic responses were obtained. There was a significant difference in the amplitude across the three groups for stimulation at 15, 20, and 30 Hz, but no difference at a stimulation of 5 and 10 Hz. Multiple comparison tests revealed that patients with SDAT had a significantly smaller amplitude than normal controls. Patients with VD had lower amplitudes than normal subjects, but this was not statistically significant. In the phase data, there were also no statistically significant differences across the three groups for any of the temporal frequencies. These results indicate that a temporal frequency deficit exists in SDAT. This phenomenon may be explained by the pathology that affects the early stage of visual signal processing.

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.

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).

This study aimed to examine the factors affecting prescription of antibiotics for acute bronchitis in ambulatory care settings in Japan.

This study investigated the decisions which Japanese paediatricians make regarding bathing a child with a common cold.