Inhibitory activity of brown algal phlorotannins against glycosidases from the viscera of the turban shell Turbo cornutus

The crude phlorotannins from the brown alga Eisenia bicyclis showed inhibitory activity against 10 of 13 kinds of glycosidases present in the viscera of the turban shell Turbo cornutus. Phloroglucinol and its oligomers – eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol and 8,8′-bieckol (hexamers), and an unidentified tetramer – were isolated from the crude phlorotannins by column and thin-layer chromatography. Phlorofucofuroeckol A, dieckol and 8,8′-bieckol inhibited α-fucosidase, β-galactosidase and β-mannosidase partially purified from T. cornutus, while phloroglucinol, eckol and the unidentified tetramer were weakly active. Dieckol was a competitive inhibitor of α-fucosidase with an inhibition constant (K _i) of 0.12?mM. The amounts of phlorotannins released after the immersion of freshly collected E. bicyclis in seawater or deionized water were estimated by high-performance liquid chromatography. Nearly all the phlorotannins were exuded into the medium following the death of the algae, whereas no phlorotannins were detected in the medium of living algae. These findings indicate that the phlorotannins deter the feeding of marine herbivorous gastropods by inhibiting the glycosidases.     

Synthesis of 3- and 21-monosulfates of [2,2,3β,4,4-²H₅]-tetrahydrocorticosteroids in the 5β-series as internal standards for mass spectrometry

The 3- and 21-monosulfates of pentadeuterated 5β-tetrahydrocorticosteroides were synthesized, starting from cortisol and 11-deoxycotisol. The principal reactions used were (1) perdeuteration of the methylene groups adjacent to the 3-oxo group of 17,20:20,21-bismethylendioxy-5β-3-ketosteroids with NaOD in CH₃OD followed by stereoselective reduction with NaBD₄, (2) sulfation of hydroxy groups with sulfur trioxide–trimethylamine complex, and (3) removal of the 17,20:20,21-bismethylendioxy group with hydrogen fluoride. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies.     

Structure of beta-1,3-xylooligosaccharides generated from Caulerpa racemosa var. laete-virens beta-1,3-xylan by the action of beta-1,3-xylanase

Recently we reported the molecular cloning and characterization of a novel beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 [Kiyohara et al. (2005) Biochem. J. 388, 949-957]. We report here the structural analysis of oligosaccharides generated from beta-1,3-xylan of a siphonous green alga, Caulerpa racemosa var. laete-virens, by the action of beta-1,3-xylanase. The enzyme degraded the polysaccharide producing oligosaccharides with different R(f)s on TLC (EX2-EX5). Sugar component, linkage, and MALDI-TOF-MS analyses revealed that EX2 and EX3 were Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,3-Xyl, respectively. On the other hand, EX4 was a mixture of Glc-1,3-Xyl-1,3-Xyl, Xyl-1,4-Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,4-Xyl, while EX5 was a mixture of tetra-saccharides containing 3-substitued Glc in addition to the same components of EX4. Branching was not likely present in EXOs prepared from the polysaccharide by the enzyme. These results strongly suggest that the C. racemosa beta-1,3-xylan is a linear heteropolysaccharide containing 1,3-Glc and 1,4-Xyl both of which are thought to be located within a beta-1,3-Xyl chain and linked via covalent bonds. This report indicates the usefulness of the enzyme for the structural analysis of beta-1,3-xylan.     

Low-volume muscular endurance and strength training during 3-week forearm immobilization was effective in preventing functional deterioration

Purpose

The purpose of this study was to determine whether endurance and strength hand grip exercises during 3-week upper limb immobilization preserve muscle oxidative capacity, endurance performance and strength.

Methods

Ten healthy adult men underwent non-dominant forearm immobilization by plaster cast for 21 days. Five healthy adult subjects were designated as the immobilization (IMM) group and five were designated as the immobilization + training (IMM+TRN) group. Grip strength, forearm circumference, dynamic handgrip endurance and muscle oxygenation response were measured before and after the 21 day immobilization period. Using near-infrared spectroscopy (NIRS), muscle oxygen consumption recovery (VO₂mus) was recorded after a submaximal exercise and the recovery time constant (TcVO₂mus) was calculated. Reactive hyperemic oxygenation recovery was evaluated after 5 minutes ischemia. Two training programs were performed by the IMM+TRN group twice a week. One exercise involved a handgrip exercise at 30% maximum voluntary contraction (MVC) at a rate of 1 repetition per 1 second until exhaustion (about 60 seconds). The other involved a handgrip exercise at 70% MVC for 2 seconds with a 2 second rest interval, repeated 10 times (40 seconds).

Results

There was a significant group-by-time interaction between the IMM and IMM+TRN groups in the TcVO₂mus (p = 0.032, F = 6.711). A significant group-by-time interaction was observed between the IMM and IMM+TRN groups in the MVC (p = 0.001, F = 30.415) and in grip endurance (p = 0.014, F = 9.791). No significant group-by-time interaction was seen in forearm circumference and reactive hyperemic oxygenation response either in IMM or IMM+TRN group.

Conclusion

The training programs during immobilization period used in this experiment were effective in preventing a decline in muscle oxidative function, endurance and strength.

     

Immunoprecipitation and MALDI-MS identification of lithocholic acid-tagged proteins in liver of bile duct-ligated rats

Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCA's known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the epsilon-amino group of lysine residues. Antibodies recognizing the 3alpha-hydroxy-5beta-steroid moiety of LCA were generated by immunizing rabbits with immunogens in which the carboxyl group of LCA was coupled to BSA via a 6-aminohexanoic acid and/or succinic acid spacer. The resulting antibodies reacted with N-alpha-(t-butoxycarbonyl)-l-lysine-epsilon-LCA, the amidated and nonamidated forms of LCA, as well as synthetically prepared LCA adducts with ovalbumin and lysozyme. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Proteins were isolated by two-dimensional electrophoresis, and their structure was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and computer-assisted programs. Proteins labeled with LCA were Rab-3, Rab-12, Rab-16, and M-Ras. Rab proteins are Ras-like small GTP binding proteins that regulate vesicle trafficking pathways. The covalent binding of the Rab proteins with LCA may influence vesicular transport or binding of vesicles to their cognate membrane and may contribute to LCA-induced liver toxicity.     

Molecular cloning and characterization of the murine gnathodiaphyseal dysplasia gene GDD1

Mutations in the GDD1 gene cause gnathodiaphyseal dysplasia, a rare human skeletal syndrome with autosomal dominant inheritance. The biochemical function(s) of GDD1 protein and the molecular pathophysiology of GDD1 mutations leading to GDD have not yet been elucidated. In this study, we characterized the complete cDNA sequence and genomic organization of the mouse GDD1 gene. Analysis of GDD1 mRNA revealed a complex alternative splicing pattern, involving five exons of the GDD1 gene. GDD1 isoforms lacking conserved amino acids at the N-terminus cytoplasmic tails, and with changes in transmembrane topology, are presumably associated with changes in protein functions and subcellular localizations of GDD1. We found GDD1 expression to be up-regulated during the course of myogenic differentiation in the murine pluripotent mesenchymal precursor cell line C2C12, whereas its expression was diminished during osteoblastic differentiation. These observations suggest diverse cellular roles of GDD1 protein.     

Analysis of estrogen receptor alpha signaling complex at the plasma membrane

There is accumulating evidence that the estrogen receptor (ER) can transduce specific signals at the plasma membrane. We tried to clarify the biological function of ER as a signaling molecule by identifying proteins that interact with the membrane-localized ER. The activation function 1 and 2 (AF-1 and AF-2) domains of ERalpha with or without the membrane-targeting sequence were stably expressed in the breast cancer cell line, MCF-7. The level of tyrosine phosphorylation of AF-2 was significantly elevated by the membrane localization. By mass-spectrometry analysis, alpha- and beta-tubulins and heat shock protein 70 were identified as the AF-1-associated proteins. Of these, tubulins are associated only with membrane-targeted AF-1.     

Tracking of V beta 8.2-positive encephalitogenic T cells by complementarity-determining region 3 spectratyping and subsequent Southern blot hybridization in Lewis rats after neuroantigen sensitization

Pathogenic T cells in organ-specific autoimmune diseases use a limited number of TCR alpha- and beta-chains. In experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by immunization with myelin basic protein, encephalitogenic T cells mainly use Vbeta8.2 TCR and clonal expansion of the Vbeta8.2 spectratype containing the EAE-specific complementarity-determining region 3 (CDR3) sequence, DSSYEQYFGPG, is found in the spinal cord throughout the course of clinical EAE. In the present study we performed temporal and spatial analyses of Vbeta8.2 spectratype expansion by CDR3 spectratyping and subsequent DNA hybridization with a probe specific for the encephalitogenic CDR3 sequence to elucidate the kinetics of encephalitogenic T cells during the induction phase after neuroantigen sensitization. It was demonstrated that Vbeta8.2 spectratype expansion and/or the positive signal in Southern blot were first detected in the regional lymph nodes as early as day 3 postimmunization and was disseminated over the lymphoid organs by day 6. Because perfusion of immunized rats with PBS erased the positive signals on day 3 postimmunization, the majority of Vbeta8.2-positive encephalitogenic T cells at the very early stage would reside within the lymphatic or blood vessels. Furthermore, removal of the draining lymph node 1, 3, and 6 days after immunization in the foot pad did not ameliorate clinical EAE. These findings strongly suggest that encephalitogenic T cells disseminate throughout the whole body very rapidly after sensitization. Analysis of pathogenic T cells at the clonal level provides useful information for designing effective immunotherapy.