Catechol O-methyltransferase homologs in Schizosaccharomyces pombe are response factors to alkaline and salt stress.

How cells of the fission yeast Schizosaccharomyces pombe respond to alkaline stress is not well understood. Here, to elucidate the molecular mechanism underlying the alkaline stress response in S. pombe, we performed DNA microarray analysis. We found that a homolog of human catechol O-methyltransferase 2 (COMT2) is highly upregulated in S. pombe cells exposed to alkaline conditions. We designated the S. pombe homolog as cmt2+ and also identified its paralog, cmt1+, in the S. pombe genome. Reverse transcription PCR confirmed that both cmt1+ and cmt2+ are upregulated within 1 h of exposure to alkaline stress and downregulated within 30 min of returning to an acidic environment. Moreover, we verified that recombinant Cmt proteins exhibit catechol O-methyltransferase activity. To further characterize the expression of cmt1+ and cmt2+, we carried out an EGFP reporter assay using their promoter sequences, which showed that both genes respond not only to alkaline but also to salt stress. Collectively, our findings indicate that the cmt promoter might be an advantageous expression system for use in S. pombe under alkaline culture conditions.

Analysis of Transglucosylation Products of Aspergillus niger α-Glucosidase that Catalyzes the Formation of α-1,2- and α-1,3-Linked Oligosaccharides.

According to whole-genome sequencing, Aspergillus niger produces multiple enzymes of glycoside hydrolases (GH) 31. Here we focus on a GH31 α-glucosidase, AgdB, from A. niger . AgdB has also previously been reported as being expressed in the yeast species, Pichia pastoris ; while the recombinant enzyme (rAgdB) has been shown to catalyze tranglycosylation via a complex mechanism. We constructed an expression system for A. niger AgdB using Aspergillus nidulans . To better elucidate the complicated mechanism employed by AgdB for transglucosylation, we also established a method to quantify glucosidic linkages in the transglucosylation products using 2D NMR spectroscopy. Results from the enzyme activity analysis indicated that the optimum temperature was 65 °C and optimum pH range was 6.0-7.0. Further, the NMR results showed that when maltose or maltopentaose served as the substrate, α-1,2-, α-1,3-, and small amount of α-1,1-ß-linked oligosaccharides are present throughout the transglucosylation products of AgdB. These results suggest that AgdB is an α-glucosidase that serves as a transglucosylase capable of effectively producing oligosaccharides with α-1,2-, α-1,3-glucosidic linkages.

A Novel α-Glucosidase of the Glycoside Hydrolase Family 31 from Aspergillus sojae.

We characterized an α-glucosidase belonging to the glycoside hydrolase family 31 from Aspergillus sojae. The α-glucosidase gene was cloned using the whole genome sequence of A. sojae, and the recombinant enzyme was expressed in Aspergillus nidulans. The enzyme was purified using affinity chromatography. The enzyme showed an optimum pH of 5.5 and was stable between pH 6.0 and 10.0. The optimum temperature was approximately 55 °C. The enzyme was stable up to 50 °C, but lost its activity at 70 °C. The enzyme acted on a broad range of maltooligosaccharides and isomaltooligosaccharides, soluble starch, and dextran, and released glucose from these substrates. When maltose was used as substrate, the enzyme catalyzed transglucosylation to produce oligosaccharides consisting of α-1,6-glucosidic linkages as the major products. The transglucosylation pattern with maltopentaose was also analyzed, indicating that the enzyme mainly produced oligosaccharides with molecular weights higher than that of maltopentaose and containing continuous α-1,6-glucosidic linkages. These results demonstrate that the enzyme is a novel α-glucosidase that acts on both maltooligosaccharides and isomaltooligosaccharides, and efficiently produces oligosaccharides containing continuous α-1,6-glucosidic linkages.

Papulonecrotic tuberculid-like eruptions after Bacille Calmette-Guerin vaccination.

One of the specific skin lesions occurring after Bacille Calmette-Guerin (BCG) vaccination is generalized tuberculid-like eruptions, which occur rarely, but have a tendency to heal spontaneously. Their pathogenesis and relationship to "true" tuberculids are poorly understood. This report presents a case of a 6-month-old girl who developed generalized papulonecrotic tuberculid-like eruptions after BCG vaccination. The skin lesions healed spontaneously in 3 months. Culture of blood, gastric juice and reverse transcription polymerase chain reaction (RT-PCR) of papulonecrotic skin biopsies were all negative for Mycobacterium tuberculosis. Histopathology of papulonecrotic eruptions revealed marked epidermal necrosis, perivascular lymphocytic infiltrates and epidermotropic infiltration of lymphocytes showing markers of CD3(+) lymphocytes (90-95% of all infiltrating cells), CD4(+) (40-50%), CD8(+) (40-50%), and CD45RO(+) (70%). In contrast, the BCG vaccination site revealed intradermal granuloma with epithelioid cells, occasional giant cells and infiltration of lymphocytes consisting of CD3(+) (60-70%), CD4(+) (40-50%), CD8(+) (30-40%), CD45RO(+) (40%), CD79a(+) (30-40%), and CD20(+) (20-30%). Our patient did not reveal any signs indicative of tuberculosis. Papulonecrotic lesions were therefore called papulonecrotic tuberculid-like eruptions, rather than tuberculids, that occurred after BCG vaccination and appeared to derive from a hypersensitive reaction mediated by immune lymphocytic infiltration.

Reconstruction method with a newly-designed bilobed flap after excision of tumours of the skin.

We have devised a bilobed skin flap for reconstruction after excision of small skin tumours. The sutured part serves as a zig-zag that leads to only slight postoperative contracture of the scar. The rotation centre of the flap is nearer to the affected area than other conventional bilobed flaps, resulting in less dog-ear deformity and distortion of tissue.

Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis.

Two types of fucan sulfate were isolated from chloroform/methanol extract of the body wall of the sea cucumber Stichopus japonicus. One type (type A) contained 3.41 mmol fucose/g and 2.35 mmol sulfate/g, and the molecular mass was determined to be 9 kDa by gel permeation chromatography (GPC). Structural analysis suggested that type A consists of a backbone of (1-->3)-linked fucosyl residues that are substituted at C-4 with fucosyl residues, and that fucosyl residues are sulfated at C-2 and/or C-4. Another type (type B) contained 3.90 mmol fucose/g and 3.07 mmol sulfate/g, and the molecular mass was determined to be 32kDa by GPC. Structural analysis showed that type B is largely composed of unbranched (1-->3)-linked fucosyl residues, and that sulfate substitution(s) occur at C-2 and/or C-4. The potential of both types to inhibit osteoclastogenesis was examined by an in vitro assay system, showing that both types of fucan sulfate inhibit osteoclastogenesis more than 95% at 50 microg/mL concentration. These results suggest that types A and B fucan sulfate from sea cucumber are potent inhibitors of osteoclastogenesis.

Basic fibroblast growth factor in an artificial dermis promotes apoptosis and inhibits expression of alpha-smooth muscle actin, leading to reduction of wound contraction.

To clarify the mechanisms underlying declines in wound contraction caused by basic fibroblast growth factor (bFGF) and the role of autologous fibroblasts in modulating wound healing, we have examined the expression of alpha-smooth muscle actin (alpha-SMA) and apoptosis in a model of wound healing using collagen sponges with and without bFGF (1 microg) and/or fibroblasts (1 x 10(6) cells/cm(2)) applied to experimentally produced full-thickness skin wounds in rats (n=10 for each group). At 7 days postoperatively, wounds filled with a fibroblast-seeded collagen sponge (fibroblast-seeded group) displayed a greater area of collagen sponge and a smaller area of fibroblasts compared with control wounds filled with collagen sponge alone (control group). Therefore, seeding of fibroblasts in the dermal substitute might retard degradation of the collagen sponge, inhibiting fibroblast infiltration into the substitute. By day 14, wounds filled with bFGF-treated collagen sponge without fibroblast seeding (bFGF group) displayed decreased alpha-SMA expression and significantly increased apoptosis compared with other wounds. Double staining revealed that apoptosis in alpha-SMA-positive fibroblastic cells was significantly increased in the bFGF group, suggesting that bFGF treatment is a potent stimulator of myofibroblast apoptosis. Furthermore, morphometric analysis demonstrated the significant decrease in the level of wound contraction and the degree of mature collagen bundle formation in the bFGF group by day 42. The bFGF group also showed increased bFGF expression in macrophages by day 28. These results suggest that bFGF administration to an artificial dermis promotes apoptosis of alpha-SMA-positive fibroblastic cells and inhibits alpha-SMA expression in the treated wound, thus reducing wound contraction.