Effects of behavioural activation on the neural basis of other perspective self-referential processing in subthreshold depression: a functional magnetic resonance imaging study.

BACKGROUND: It has been demonstrated that negatively distorted self-referential processing, in which individuals evaluate one's own self, is a pathogenic mechanism in subthreshold depression that has a considerable impact on the quality of life and carries an elevated risk of developing major depression. Behavioural activation (BA) is an effective intervention for depression, including subthreshold depression. However, brain mechanisms underlying BA are not fully understood. We sought to examine the effect of BA on neural activation during other perspective self-referential processing in subthreshold depression. METHOD: A total of 56 subjects underwent functional magnetic resonance imaging scans during a self-referential task with two viewpoints (self/other) and two emotional valences (positive/negative) on two occasions. Between scans, while the intervention group (n = 27) received BA therapy, the control group (n = 29) did not. RESULTS: The intervention group showed improvement in depressive symptoms, increased activation in the dorsal medial prefrontal cortex (dmPFC), and increased reaction times during other perspective self-referential processing for positive words after the intervention. Also, there was a positive correlation between increased activation in the dmPFC and improvement of depressive symptoms. Additionally, there was a positive correlation between improvement of depressive symptoms and increased reaction times. CONCLUSIONS: BA increased dmPFC activation during other perspective self-referential processing with improvement of depressive symptoms and increased reaction times which were associated with improvement of self-monitoring function. Our results suggest that BA improved depressive symptoms and objective monitoring function for subthreshold depression.

Demonstration of an endo-beta-galactosidase and an endo-beta-xylosidase that degrade the proteoglycan linkage region.

Purified proteochondroitin sulfate was incubated at pH 4.0 with a rabbit liver lysosomal enzyme fraction. The formed degradation products were isolated by gel filtration followed by reduction with NaB3H4. After acid hydrolysis of the reduced digest, the hydrolysate was passed through Dowex 50W-X8 and 1-X2 columns. The separated neutral sugars were then subjected to paper chromatography, which demonstrated that galactose and xylose had been at the reducing termini of chondroitin sulfate after incubation with the liver lysosomal preparation. These results suggest the presence in the liver lysosomal preparation of an endo-beta-galactosidase and an endo-beta-xylosidase which act at the linkage region of proteochondroitin sulfate.

Hyaluronate synthesized by cultured skin fibroblasts derived from patients with Werner's syndrome.

Hyaluronate in cultured skin fibroblasts derived from patients with Werner's syndrome, who excrete large amounts of urinary hyaluronate, was investigated. The amount of hyaluronate secreted into the medium by Werner's fibroblasts was 2-3-times that of normal fibroblasts, whereas no difference in enzyme activities related to the degradation of hyaluronate was found. Werner's fibroblasts were then cultured in the presence of [3H]glucosamine, and the amount of [3H]hyaluronate and its chain lengths in the medium and matrix (trypsinate) fractions were compared with those of normal cells. No significant difference in the chain length of hyaluronate was observed between normal and Werner's fibroblasts. On the other hand, a significant increase of hyaluronate was found in the matrix fraction of Werner's fibroblasts when the cells reached confluency. In addition, a hyaluronate of small chain length was found in the matrix fraction of Werner's fibroblasts, although this was absent from that of normal cells. It was concluded that the constituents of the extracellular matrix of Werner's fibroblasts differed from those of normal cells, characterized by the presence of a large amount of hyaluronate and a relatively small hyaluronate chain.

Construction of high, intermediate and low-copy-number promoter-probe plasmids for Bacillus subtilis.

We constructed promoter-probe plasmids for Bacillus subtilis based on the promoterless gene for penicillinase (penP) of Bacillus licheniformis. A Bacillus stearothermophilus DNA fragment, which contained translational stop codons for all three reading frames and transcription terminators of the alpha-amylase gene, was inserted upstream from the Shine-Dalgarno sequence for the penP gene. A low-copy-number plasmid (about 10 copies per chromosome), pPF101, carried tetracycline-resistance gene, whereas another high-copy-number plasmid (about 55 copies per chromosome), pPF201, conferred resistance to kanamycin. The intermediate-copy-number plasmids (about 20 copies per chromosome), pPF001, pPF002, pPF011 and pPF012, conferred chloramphenicol resistance to both B. subtilis and Escherichia coli. Vector plasmids, pPF011 and pPF012, were provided with single cloning sites for BamHI, SacI, SmaI and XbaI upstream from the protein coding region of penP, whereas other plasmids contained only unique BamHI cloning sites. The applicability of these plasmids was demonstrated by cloning a promoter for the alpha-amylase gene.

Demonstration of N-acetylchondrosine-degrading beta-glucuronidase in rabbit liver.

N-Acetylchondrosine was incubated at pH 4.0 with a rabbit-liver crude enzyme extract. Gel filtration of the reaction products on Sephadex G-15 revealed the presence of monosaccharide liberated from the disaccharide. The monosaccharide fraction was analyzed by gas-liquid chromatography, and identified as a mixture of glucuronic acid and N-acetylgalactosamine. These results indicate the presence of beta-glucuronidase, which degrades N-acetylchondrosine, in rabbit liver. The discovery of the presence of this enzyme may help to establish the complete degradation process of chondroitin sulfates.

Demonstration of chondroitin sulfates degrading endo-beta-glucuronidase activity in rabbit liver.

Reduced chondroitin sulfate was incubated with rabbit liver extracts followed by reduction once more with sodium [3H]borohydride, and then passed through a Sephadex G-100 column. Chondroitin sulfate obtained from the incubation medium at pH 4 was only slightly depolymerized and was highly radioactive. Paper chromatographic analyses showed that glucuronic acid residues became exposed at the reducing terminal of chondroitin sulfate after incubation with the liver extracts. These results suggest that endo-beta-glucuronidase activity which degrades chondroitin sulfate is present in the rabbit liver.

Regulation of hyaluronate metabolism by progesterone in cultured fibroblasts from the human uterine cervix.

The effects of progesterone, dehydroepiandrosterone sulfate and 17beta-estradiol on the synthesis and degradation of hyaluronate were investigated using human uterine cervix fibroblasts. The cells were incubated with [3H]glucosamine in the presence of the hormones and then [3H]hyaluronate was isolated from the medium. The changes in the radioactivity of [3H]hyaluronate showed that progesterone suppressed hyaluronate synthesis by 22% of the control levels, while dehydroepiandrosterone sulfate and 17beta-estradiol enhanced it by 22% and 12% of the control levels, respectively. Furthermore, progesterone induced degradation of high-molecular-weight [14C]hyaluronate into low-molecular-weight hyaluronate (Mr approximately 40000). These results suggest that in cultured fibroblasts from the human uterine cervix progesterone converts hyaluronate metabolism from the synthesis phase to the degradation phase.

Cloning, functional expression and tissue distribution of human alpha 1c-adrenoceptor splice variants.

We report the cloning and characterization of two isoforms of human alpha 1c-adrenoceptor cDNA (alpha 1c-2, alpha 1c-3). These isoforms are generated by alternative splicing and differ from the clone we previously isolated (alpha 1c-1) in their length and sequences of the C-terminal domain. Tissue distribution of mRNAs showed that these variants co-express with alpha 1c-1 in the human heart, liver, cerebellum and cerebrum. Despite the structural differences, functional experiments in transfected CHO cells showed that the three isoforms have similar ligand binding properties, and all couple with phospholipase C/Ca2+ signaling pathway.

Hyaluronate depolymerization activity induced by progesterone in cultured fibroblasts derived from human uterine cervix.

High-molecular-weight [14C]hyaluronate was incubated with cultured fibroblasts from human uterine cervix and skin, and then the depolymerization of the hyaluronate was investigated. [14C]Hyaluronate in the medium of skin fibroblasts was depolymerized into a constant molecular weight (M(r) about 40,000), whereas that of cervix fibroblasts was not depolymerized, irrespective of incubation period. However, when progesterone was added to the medium of cervix fibroblasts, hyaluronate was depolymerized to the same extent as that in skin fibroblasts. The reducing terminal sugar of the depolymerized hyaluronate was N-acetylglucosamine. These results suggest that a hyaluronate-depolymerizing enzyme, endo-beta-N-acetylglucosaminidase, was induced by progesterone in cultured fibroblasts derived from human uterine cervix.

Effect of monensin on the synthesis of beta-D-xyloside-initiated glycosaminoglycan and its linkage region oligosaccharides in human skin fibroblasts.

Human skin fibroblasts were cultured with a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside (Xyl-MU) as an initiator, and the effects of monensin, which destroys the normal structure of the Golgi complex, on the synthesis of Xyl-MU-initiated glycosaminoglycan (GAG-MU) and its linkage region oligosaccharides were investigated. When the cells were incubated with Xyl-MU in the presence of monensin, the synthesis of GAG-MU was inhibited. In addition, the synthesis of Galbeta1-3Galbeta1-4Xylbeta1-MU as an intermediate of GAG-MU was inhibited, whereas the synthesis of Galbeta1-4Xylbeta1-MU, which is formed prior to Galbeta1-3Galbeta1-4Xylbeta1-MU, was not. These results indicate that inhibition of GAG-MU synthesis by monensin occurs at the point where the second galactose is joined to Galbeta1-4Xylbeta1-MU.

Characterization of beta-D-xyloside-induced glycosaminoglycans and oligosaccharides in cultured human skin fibroblasts.

Human skin fibroblasts were incubated in the presence of a fluorogenic xyloside, 4-methylumbelliferyl beta-D-xyloside. Three fluorogenic components were isolated and purified from the culture medium by gel permeation high-performance liquid chromatography. Their structures were then characterized by enzymatic digestion, fast-atom-bombardment mass spectrometry, gas-liquid chromatography, and electrophoresis on cellulose acetate membrane. The results showed that one of the components was a mixture of dermatan sulfate (70%) and chondroitin sulfate (30%), bearing the 4-methylumbelliferone at the reducing termini, and having an average molecular weight of 9,200. The others had the structures galactosyl-galactosyl-xylosyl-4-methylumbelliferone and galactosyl-xylosyl-4-methylumbelliferone, respectively, representing the linkage region between the glycosaminoglycan chains and core protein, except that 4-methylumbelliferone replaced the amino acid. Moreover, it was demonstrated that these oligosaccharides were intermediates of glycosaminoglycan synthesis, not depolymerized products.

Application of 2-aminopyridine fluorescence labeling to glycosaminoglycans.

A pyridylamination method was applied to glycosaminoglycans and the characteristics of the resulting pyridylamino glycosaminoglycans were examined. First, glycosaminoglycan chains, which uniformly possess a xylose residue at their reducing termini, were liberated from proteoglycan by successive digestion with protease and endo-beta-xylosidase. Then the glycosaminoglycan chains were coupled with 2-aminopyridine by reductive amination with sodium cyanoborohydride for 15 h according to the method of Hase, S. et al. [J. Biochem. 95, 197-203 (1984)]. The pyridylamination reaction caused neither depolymerization, de-N-acetylation, nor de-N- or de-O-sulfation. The pyridylamino glycosaminoglycan chains had an intact linkage region (GlcA-Gal-Gal-Xyl) between the carbohydrate chain and the peptide core of the proteoglycan. These pyridylamino glycosaminoglycans should be useful as substrates for endo-type glycosidases that act on glycosaminoglycan chains and as markers for studies of glycosaminoglycan metabolism.

Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase.

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.

Characterization and biological significance of sialyl alpha 2-3galactosyl beta 1-4xylosyl beta 1-(4-methylumbelliferone) synthesized in cultured human skin fibroblasts.

Human skin fibroblasts were incubated in the presence of a fluorogenic xyloside, 4-methyl-umbelliferyl-beta-D-xyloside (Xyl-MU), then the cultured medium was recovered, concentrated with a lyophilizer, and dialyzed against distilled water. The structures of the Xyl-MU derivatives purified from the dialyzable fraction were investigated. In addition to established glycosaminoglycans-MU (GAGs-MU), Gal-Gal-Xyl-MU, Gal-Xyl-MU, sulphate-GlcA-Xyl-MU, GlcA-Xyl-MU, and Xyl-Xyl-MU, which were induced by Xyl-MU, an oligosaccharide having fluorescence was purified using a combination of gel filtration, ion-exchange chromatography and high-performance liquid chromatography, then subjected to carbohydrate composition analysis, enzyme digestion, Smith degradation, 1H-NMR, and ion-spray mass spectrometric analysis. From the data obtained, the oligosaccharide was considered to have the structure SA alpha 2-3Gal beta 1-4Xyl beta 1-MU. The amount of MU-oligosaccharide in the cell culture increased with time and was dependent on the amount of Xyl-MU added. Its production was also different from that of Gal-Gal-Xyl-MU and Gal-Xyl-MU, which are biosynthetic intermediates of GAG-MU. Addition of CDP, an inhibitor of sialytransferase, to the cell culture medium increased the secretion of GAG-MU. These results suggest that SA-Gal-Xyl-MU production may be related to the regulation of GAG-MU biosynthesis.

A novel oligosaccharide, xylosyl beta 1-4xylosyl beta 1-(4-methylumbelliferone), synthesized by cultured human skin fibroblasts in the presence of 4-methylumbelliferyl-beta-D-xyloside.

4-Methylumbelliferyl-beta-D-xyloside (Xyl-MU) was added to the medium of cultured human skin fibroblasts. After incubation, the culture medium was pooled, concentrated with a lyophilizer, and dialyzed against distilled water. Then the Xyl-MU derivatives in the diffusate were purified by gel-filtration and HPLC. A novel Xyl-MU derivative was obtained, in addition to the previously reported Xyl-MU derivatives, Xyl-MU-induced glycosaminoglycan (GAG-MU), SA-Gal-Xyl-MU, GlcA-Xyl-MU, Gal-Gal-Xyl-MU, and Gal-Xyl-MU. This Xyl-MU derivative was subjected to carbohydrate composition analysis, enzyme digestion, Smith degradation and ion-spray mass spectrometric analysis, and the results indicated that it was Xyl beta 1-4Xyl beta 1-MU. Although the quantity of Xyl beta 1-4Xyl beta 1-MU synthesized by human skin fibroblasts increased with incubation time, its production was independent of that of the GAG-MU. Xyl-Xyl-MU is different from the intermediates in the regular pathway of GAG-MU biosynthesis initiated by added Xyl-MU, posing an interesting question as to its significance in GAG biosynthesis.

Preparation and application of a fluorogenic substrate for endo-beta-xylosidase.

The present paper describes a fluorometric assay for galactosaminoglycan-degrading endo-beta-xylosidase, utilizing glycosaminoglycan chains bearing a 4-methylumbelliferyl group at the reducing terminus as a substrate. This fluorogenic substrate is synthesized by human skin fibroblasts cultured in the presence of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside. The assay is based on measurement of the fluorescence of 4-methylumbelliferone, enzymatically liberated from the synthetic substrate by endo-beta-xylosidase. We examined the applicability of the assay for analysis of endo-beta-xylosidase activity.

The application of the Milner-Avigad method for the quantitative determination of endouronidase activities.

The Milner-Avigad method (Milner, Y. and Avigad, G. (1967) Carbohydr. Res. 4, 359-361) has been shown to have high specificity for measuring the reducing powers of free hexuronic acids; its applicability in the quantitative determination of endouronidase activity was the purpose of this investigation. Among the constituent monosaccharides of glycosaminoglycans, hexuronic acids showed high color yield by this method, while xylose, galactose and N-acetylhexosamine recorded negligible color yield. Among the monosaccharide residues at the reducing terminals of oligosaccharides, only hexuronic acids exhibited color yield. However, the color yield was less than that of free hexuronic acids. Gel filtration chromatography of reaction products revealed that the cleavage of the oligosaccharide chains and the resultant exposure of new reducing terminals were not caused by the reaction procedures involved in this method. These data indicate that the Milner-Avigad method is useful for determining the presence of hexuronic acid residues preferentially at reducing terminals of glycosaminoglycan moieties. Thus, it supported the conclusion that the Milner-Avigad method is applicable for the quantitative determination of endouronidase activity using glycosaminoglycan as a substrate.

Determination of the reducing terminal sugars of glycosaminoglycans using 2-aminopyridine.

The fluorescence labeling method (Takemoto, H. et al. (1985) Anal. Biochem. 145, 245-250) has been shown to have high sensitivity for measuring the sugar composition of glycoproteins. In the present study, its applicability for analysis of the reducing terminal sugars of glycosaminoglycans was investigated. The procedure involved coupling of glycosaminoglycans with 2-aminopyridine, followed by hydrolysis and N-acetylation, and then analysis by high-performance liquid chromatography on a reverse-phase column. The method was found to be useful for simultaneous determination of acidic, neutral and amino sugars at the reducing termini of glycosaminoglycan moieties.

Analysis of glycosaminoglycans by high-performance liquid chromatography.

Glycosaminoglycan chains were liberated from proteoglycan by successive digestion with protease and endo-beta-xylosidase. The glycosaminoglycan chains were then labeled with a fluorescent reagent, 2-aminopyridine, by reductive amination. The resulting pyridylamino-glycosaminoglycans, including hyaluronic acid, heparan sulfate, chondroitin sulfate/dermatan sulfate and heparin, were separated by ion-exchange high-performance liquid chromatography using a TSK gel SAX analytical column with a limit of sensitivity in the picomol range. With the combined use of a dermatan sulfate-degrading enzyme, chondroitinase B, chondroitin sulfate and dermatan sulfate were identified and quantified, separately. About 50 mg of wet animal tissue was enough for analysis of each glycosaminoglycan with satisfactory results. This method facilitates rapid separation and microanalysis of glycosaminoglycans.

Simple measurement of glycosaminoglycan produced by cultured fibroblasts using 4-methylumbelliferyl beta-D-xyloside.

A simple and rapid method was devised for measurement of glycosaminoglycan produced by cultured cells. 4-Methylumbelliferyl-beta-D-xyloside was added to the medium of the cultured cells. After incubation, glycosaminoglycan, which was produced from 4-methylumbelliferyl-beta-D-xyloside as a primer and secreted into the medium, was separated by proteinase digestion, trichloroacetic acid treatment and ethanol precipitation. The glycosaminoglycan, bearing a fluorescent moiety at the reducing terminal, was electrophoresed on cellulose acetate membrane, and then the fluorescent band visible on the membrane was extracted. The fluorescence of the band was measured, and from this the amount of glycosaminoglycan was estimated. Using this method, it was possible to quantify a very small amount of glycosaminoglycan with relatively high sensitivity without employing a radioisotope. This method was applied for determination of glycosaminoglycan produced by cultured fibroblasts from human uterine cervix, and also the effect of a hormone on glycosaminoglycan production. It was found that uterine cervical fibroblasts produced twice as much glycosaminoglycan as skin fibroblasts.