Genetic analysis and characterization of poly(aspartic acid) hydrolase-1 from Sphingomonas sp. KT-1

Sphingomonas sp. KT-1 hydrolyzes poly(aspartic acid) (PAA) containing alpha- and beta-amide units and has at least two different types of PAA hydrolases. The PAA hydrolase-1 hydrolyzes selectively beta-beta amide units in PAA. Molecular cloning of PAA hydrolase-1 from Sphingomonas sp. KT-1 has been carried out to characterize its gene products. Genetic analysis shows that the deduced amino acid sequence of PAA hydrolase-1 has a similarity with those of the catalytic domain of poly(3-hydroxybutyric acid) (PHB) depolymerases from Alcaligenes faecalis AE122 and Pseudomonas lemoignei. Site-specific mutation analysis indicates that (176)Ser is a part of a strictly conserved pentapeptide sequence (Gly-Xaa-Ser-Xaa-Gly), which is the lipase box, and plays as an active residue.     

Variations in plasma melatonin levels of the rainbow trout (Oncorhynchus mykiss) under various light and temperature conditions

Daily variations in plasma melatonin levels in the rainbow trout Oncorhynchus mykiss were studied under various light and temperature conditions. Plasma melatonin levels were higher at mid-dark than those at mid-light under light-dark (LD) cycles. An acute exposure to darkness (2 hr) during the light phase significantly elevated the plasma melatonin to the level that is comparable with those at mid-dark, while an acute exposure to a light pulse (2 hr) during the dark phase significantly suppressed melatonin to the level that is comparable with those at mid-light. Plasma melatonin kept constantly high and low levels under constant darkness and constant light, respectively. No circadian rhythm was seen under both conditions. When the fish were subjected to simulative seasonal conditions (simulative (S)-spring: under LD 13.1:10.9 at 13 degrees C; S-summer: under LD 14.3:9.7 at 16.5 degrees C; S-autumn: under LD 11.3:12.7 at 13 degrees C; S-winter: under LD 10.1:13.9 at 9 degrees C), melatonin levels during the dark phase were significantly higher than those during the light phase irrespective of simulative seasons. The peak melatonin level in each simulative season significantly correlated with temperature but not with the length of the dark phase employed. In addition, the peak melatonin level in S-autumn was significantly higher than those in S-spring although water temperature was the same under these conditions. These results indicate that the melatonin rhythm in the trout plasma is not regulated by an endogenous circadian clock but by combination of photoperiod and water temperature.     

Cloning and expression of beta1,4-galactosyltransferase gene from Helicobacter pylori

Helicobacter pylori, which is a human pathogen associated with gastric and duodenal ulcer, has been shown to express human oncofetal antigens Lewis X and Lewis Y. Although the mammalian glycosyltransferases that synthesize these structures are well characterized, little is known about the corresponding bacterial enzymes. We report that a novel beta1,4-galactosyltransferase gene (HpgalT) involved in the biosynthesis of lipopolysaccharides in H. pylori has been cloned and expressed in Escherichia coli. The deduced amino acid sequence of the protein (HpGal-T) encoded by HpgalT consists of 274 residues with the calculated molecular mass of 31,731 Da, which does not show significant similarity to those of beta1,4-galactosyltransferases from mammalian sources and Neisseria It was confirmed that HpGal-T catalyzed the introduction of galactose from UDP-Gal in a beta1,4 linkage to accepting N-acetylglucosamine (GlcNAc) residues by means of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). When the E.coli cells which overexpressed HpgalT was coupled with the UDP-Gal production system, which consisted of recombinant E.coli cells overexpressing its UDP-Gal biosynthetic genes and Corynebacterium ammoniagenes, N-acetyllactosamine, a core structure of lipopolysaccharide of H.pylori, was efficiently produced from orotic acid, galactose, and GlcNAc.     

High expression of glycogen-debranching enzyme in Escherichia coli and its competent purification method

Glycogen-debranching enzyme (GDE) gene from Saccharomyces cerevisiae was cloned and expressed into Escherichia coli. A 99.3% homology was found between the nucleotide sequences of GDE gene harbored in the recombinant E. coli plasmid (pTrc99A) and the open reading frame (902039-906646 position) of the 4608-bp fragment of S. cerevisiae chromosome XVI. We investigated the best conditions for GDE expression. When the cultivation temperature of recombinant E. coli strains was lowered to 25 degrees C and the isopropyl-beta-d-thiogalactopyranoside (IPTG) concentration used for induction was decreased to as low as 0.02 mM, a total of about 33 mg of recombinant GDE can be isolated from a liter culture as estimated by amylo-1,6-glucosidase activity. Consecutively, we developed a new method for purifying GDE. The method requires only a single-step purification via beta-cyclodextrin-immobilized Sepharose 6B (beta-CD Sepharose 6B) affinity chromatography and renders a 90% recovery of the enzyme. Moreover, the purified recombinant GDE is a homogeneous protein and possesses the same characteristics as those of S. cerevisiae. With the highly expressed GDE in recombinant E. coli and a rapid and effective purification method, we successfully resolved the hurdle always faced for obtaining an ample amount of purified GDE. The availability of GDE, hence, may allow advancement on GDE studies and provide new prospects for GDE on biotechnological application.     

Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs

Hedgehog (HH) and Decapentaplegic (DPP) direct anteroposterior patterning in the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. Here, we show that the activity of DPP is graded and is directly regulated by a novel HH-dependent mechanism. DPP activity was monitored by visualizing the activated form of Mothers against dpp (MAD), a cytoplasmic transducer of DPP signaling. We found that activated MAD levels are highest near the source of DPP but are unexpectedly low in the cells that express dpp. HH induces dpp in these cells; it also attenuates their response to DPP by downregulating expression of the DPP receptor thick veins (tkv). We suggest that regulation of tkv by HH is a key part of the mechanism that controls the level and distribution of DPP.     

Y-27632 inhibits gastric motility in conscious rats

Y-27632, a highly selective inhibitor of p160ROCK, desensitizes the smooth muscle to Ca2+ and inhibits smooth muscle contraction. While this drug has the potential to become a novel drug for hypertension, it might also affect other smooth muscle, including that of gastrointestinal tract. We studied the effects of Y-27632 on gastric contractions in conscious rats. Strain gauge force transducers were sutured onto the serosal side of the gastric antrum and contractions were recorded before and after the intravenous injection of Y-27632. Doses of 1.0 mg/kg to 10 mg/kg significantly decreased contraction amplitude and the motility index in a dose dependent manner. With 10 mg/kg, the mean amplitude was decreased by up to 69 +/- 14% and the motility index by up to 81 +/- 7%. The change occurred immediately after drug infusion and lasted for 3.5h. Contraction frequency showed only a slight decrease. No signs of bowel obstruction were observed. These results indicate that Rho-mediated Ca sensitization has a role in the physiologic contractions of gastric smooth muscle in rats. Y-27632 is useful to investigate the physiology of gastrointestinal motility.     

A Physical Map of Arabidopsis thaliana Chromosome 3 Represented by Two Contigs of CIC YAC, P1, TAC and BAC Clones

We have constructed a physical map of Arabidopsis thaliana chromosome3 by ordering the clones from CIC YAC, P1, TAC and BAC librariesusing the sequences of a variety of genetic and EST markersand terminal sequences of clones. The markers used were 112DNA markers, 145 YAC end sequences, and 156 end sequences ofP1, TAC and BAC clones. The entire genome of chromosome 3, exceptfor the centromeric and telomeric regions, was covered by twolarge contigs, 13.6 Mb and 9.2 Mb long. This physical map willfacilitate map-based cloning experiments as well as genome sequencingof chromosome 3. The map and end sequence information are availableon the KAOS (Kazusa Arabidopsis data Opening Site) web siteat http://www.kazusa.or.jp/arabi/.     

Structural Analysis ofArabidopsis thaliana Chromosome 5. VI. Sequence Features of the Regions of 1,367,185 bp Covered by 19 Physically Assigned P1 and TAC Clones

Nineteen Pl and TAC clones, which have been mapped on the finephysical map of the Arabidopsis thaliana chromosome 5, weresequenced according to the shotgun-based strategy, and theirstructural features were analysed. The total length of the regionssequenced in this study was 1,367,185 bp. Combining this withthe regions covered by 90 P1 and TAC clones proviously reported,the total length of chromosome 5 sequenced to date becomes 8,058,855bp. On the basis of similarity search against protein and ESTdatabases and gene modeling with computer programs, a totalof 330 potential protein-coding regions were identified, bringingan average density of the genes to approximately one gene per4.1 kb. Introns were identified in 81.0% of the potential proteingenes for which the entire gene structure was predicted, withan average number per gene of 4.2 and an average length of theintrons of 180 bp. The RNA-coding genes identified were 9 tRNAgenes corresponding to 8 amino acid species and 2 genes forU2 nuclear RNA. These sequence features are essentially identicalto those in the previously reported sequences. The sequencedata and gene information are available on the World Wide Webdatabase KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

A Novel Phthalimide Derivative,TC11, Has Preclinical Effects on High-Risk Myeloma Cells and Osteoclasts

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the C_max was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.     

Effects of Acute Endurance Exercise Performed in the Morning and Evening on Inflammatory Cytokine and Metabolic Hormone Responses

Purpose

To compare the effects of endurance exercise performed in the morning and evening on inflammatory cytokine responses in young men.

Methods

Fourteen healthy male participants aged 24.3 ± 0.8 years (mean ± standard error) performed endurance exercise in the morning (0900–1000 h) on one day and then in the evening (1700–1800 h) on another day with an interval of at least 1 week between each trial. In both the morning and evening trials, the participants walked for 60 minutes at approximately 60% of the maximal oxygen uptake (V·O2max) on a treadmill. Blood samples were collected to determine hormones and inflammatory cytokines at pre-exercise, immediately post exercise, and 2 h post exercise.

Results

Plasma interleukin (IL)-6 and adrenaline concentrations were significantly higher immediately after exercise in the evening trial than in the morning trial (P < 0.01, both). Serum free fatty acids concentrations were significantly higher in the evening trial than in the morning trial at 2 h after exercise (P < 0.05). Furthermore, a significant correlation was observed between the levels of IL-6 immediately post-exercise and free fatty acids 2 h post-exercise in the evening (r = 0.68, P < 0.01).

Conclusions

These findings suggest that the effect of acute endurance exercise in the evening enhances the plasma IL-6 and adrenaline concentrations compared to that in the morning. In addition, IL-6 was involved in increasing free fatty acids, suggesting that the evening is more effective for exercise-induced lipolysis compared with the morning.