[Polycythemia vera diagnosis during follow-up in a case of extrahepatic portal obstruction with portal biliopathy].

We describe the case of a 60-year-old man who presented at our hospital with abdominal pain and elevated hepatobiliary enzymes. Computed tomography showed portal thrombosis and cavernous transformation as well as increased wall thickness and a stricture in the biliary tract. At that time, the cause of the portal thrombosis was unknown. During follow-up, the blood cell counts (WBCs and platelets) were remarkably increased, and a test performed for the JAK2V617F mutation was positive. We diagnosed the patient with polycythemia vera. Our findings demonstrate that a patient presenting with portal thrombosis, portal biliopathy, and underlying myeloproliferative neoplasms should be carefully examined, even in the absence of the typical blood alterations.

[A case of bullous systemic lupus erythematosus with sloughing esophagitis].

A 57-year-old female presented with a chief complaint of odynophagia during medical treatment for systemic lupus erythematosus (SLE). Endoscopy revealed sloughed mucosa along the entire esophageal length, and normal mucosa was easily stripped by withdrawal of the biopsy forceps. Blistering eruptions subsequently appeared on her upper extremities, trunk, and oral cavity. Direct immunofluorescence of a skin biopsy specimen demonstrated linear deposits of IgG, IgM, and complement at the dermoepidermal junction. On the basis of these findings, a diagnosis of bullous SLE was made. This autoimmune blistering disease can occur in the course of SLE and is rarely accompanied by sloughing of the esophageal mucosa.

A novel dual lock method for down-regulation of genes, in which a target mRNA is captured at 2 independent positions by linked locked nucleic acid antisense oligonucleotides.

Nuclear factor κB (NFκB), which is composed of the RelA and p50 subunits, binds to NFκB response elements (NREs) and stimulates the transcription of inflammation-related genes. Here, locked nucleic acid (LNA) antisense oligonucleotides (ASOs) complementary to the termini of the 3'- and 5'-untranslated regions (UTRs) of the RelA mRNA were generated; these molecules were named 3'-LNA and 5'-LNA, respectively. To evaluate their effects on NFκB activity, HeLa cells were co-transfected with the LNA ASOs and a luciferase reporter gene carrying an NRE. Transfection of the cells with 3'-LNA reduced NFκB activity by 30-40%, without affecting RelA mRNA accumulation. Concomitant transfection of HeLa cells with 5'-LNA and 3'-LNA resulted in a 70% reduction in NFκB activity. Furthermore, partial poly(A) tail shortening occurred in LNA ASO-transfected cells. We also employed triethylene glycol as a spacer to link 5'-LNA and 3'-LNA. Reporter gene assays showed that the spacer-linked LNA ASO reduced NFκB activity similarly to a combination of 5'-LNA and 3'-LNA. In addition, an in vitro translation assay revealed that spacer-linked LNA ASOs inhibited the translation of a target mRNA in a specific manner. In summary, this study describes a novel antisense method capturing the target mRNA at independent positions.

Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes.

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of ß-1,3-galactan main chains and ß-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-ß-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed ß-1,3-galactan, but did not act on ß-1,3-glucan, ß-1,3:1,4-glucan, xyloglucan, and agarose. It released various ß-1,3-galactooligosaccharides together with Gal from ß-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on ß-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-ß-1,3-glucanase and endo-ß-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-ß-1,3-galactanase.

Bifunctional cytosolic UDP-glucose 4-epimerases catalyse the interconversion between UDP-D-xylose and UDP-L-arabinose in plants.

UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The N-terminal amino acid sequence of the enzyme revealed that it is encoded by a predicted UDP-Glc 4-epimerase gene, PsUGE1, and is distinct from the UDP-Xyl 4-epimerase localized in the Golgi apparatus. rPsUGE1 (recombinant P. sativum UGE1) expressed in Escherichia coli exhibited both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities with apparent Km values of 0.31, 0.29, 0.16 and 0.15 mM for UDP-Glc, UDP-Gal (UDP-galactose), UDP-Ara (UDP-L-arabinose) and UDP-Xyl respectively. The apparent equilibrium constant for UDP-Ara formation from UDP-Xyl was 0.89, whereas that for UDP-Gal formation from UDP-Glc was 0.24. Phylogenetic analysis revealed that PsUGE1 forms a group with Arabidopsis UDP-Glc 4-epimerases, AtUGE1 and AtUGE3, apart from a group including AtUGE2, AtUGE4 and AtUGE5. Similar to rPsUGE1, recombinant AtUGE1 and AtUGE3 expressed in E. coli showed high UDP-Xyl 4-epimerase activity in addition to their UDP-Glc 4-epimerase activity. Our results suggest that PsUGE1 and its close homologues catalyse the interconversion between UDP-Xyl and UDP-Ara as the last step in the cytosolic de novo pathway for UDP-Ara generation. Alternatively, the net flux of metabolites may be from UDP-Ara to UDP-Xyl as part of the salvage pathway for Ara.

Molecular cloning and expression in Pichia pastoris of a Irpex lacteus exo-beta-(1-->3)-galactanase gene.

A gene encoding exo-beta-(1-->3)-galactanase from Irpex lacteus was cloned by reverse transcriptase-PCR. The deduced amino acid sequence showed high similarity with exo-beta-(1-->3)-galactanases from other sources. The molecular mass of the mature form was calculated to be 45,520 Da. The gene product expressed in Pichia pastoris specifically hydrolyzed beta-(1-->3)-galactooligosaccharides, as did other exo-beta-(1-->3)-galactanases. The recombinant enzyme showed high activity toward arabinogalactan-proteins (AGPs) from radish as well as beta-(1-->3)-galactan. Product analysis revealed that the enzyme released beta-(1-->6)-galactobiose, beta-(1-->6)-galactotriose, and alpha-L-arabinofuranosyl-(1-->3)-beta-galactosyl-(1-->6)-galactose together with Gal from beta-(1-->3)-galactans attached with and without beta-(1-->6)-galactosyl branches prepared from acacia gum. These results indicate that the exo-beta-(1-->3)-galactanase from I. lacteus efficiently hydrolyzes beta-(1-->3)-galactan main chains of AGPs by bypassing beta-(1-->6)-galactosyl side chains.

A murine model of atopic dermatitis can be generated by painting the dorsal skin with hapten twice 14 days apart.

Drug development involves pharmacometric experiments in animals. Such experiments should limit animal pain and stress. Conventional murine models of atopic dermatitis (AD) used in drug development are generated by weekly painting of hapten on dorsal skin for 5 weeks. The present study aimed to develop a protocol that involves less animal distress. The experiments focused on serum total IgE levels, which are a marker of AD. The conventional protocol induced ever rising IgE levels. Experiments with extended intervals between sensitizations showed that IgE peaked ~5 days after the second sensitization, after which it returned to the control level within 12-19 days. An additional third sensitization on day 28 further increased the serum IgE level. In the 4-5 days after the second sensitization, the dorsal skin exhibited typical AD-like lesions with edema, scabs, epithelial-cell hypertrophy, marked mast-cell and lymphocyte infiltration of dermis, and increased IL-4, IL-6, IL-10, IL-1ß, IL-17A, IFN-γ and TNF-α expression. Thus, two 2,4-dinitrofluorobenzene sensitizations yield a murine AD model in less than 20 days. This study shows that animal model protocols used in drug development can be fine-tuned so that they remain effective yet cause animals less stress and pain.

Degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from Neurospora crassa.

Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have ß-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an α-L-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-ß-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-L-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on ß-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, ß-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.