Interactions of arsenic with human metallothionein-2

Arsenic is a toxic element that is found in the atmosphere, as well as in aquatic and terrestrial environments. We have demonstrated that As(3+) binds to human metallothionein-2 (hMT-2) by UV absorption spectroscopy, inductively coupled plasma-atomic emission spectrometry (ICP-AES), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-TOF-MS revealed that the structure of the adduct formed by arsenic and hMT-2 (As-hMT-2) was not homogeneous. The maximum molar ratio of arsenic to hMT-2 was found to be more than 6:1 on ICP-AES, UV absorption spectroscopy and MALDI-TOF-MS. The ratio of the number of sulfhydryl groups in hMT-2 that bound arsenic was 3:1, which is the same as the ratios reported previously for arsenic-glutathione and arsenic-phytochelatin complexes.     

Constitutive OX40/OX40 ligand interaction induces autoimmune-like diseases

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4(+) T cells, but not CD8(+) T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4(+) T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4(+) T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity.     

Photosynthesis–nitrogen relationships in species at different altitudes on Mount Kinabalu, Malaysia

In situ photosynthetic nitrogen-use efficiency (PNUE, photosynthetic capacity per unit leaf nitrogen) was investigated in species that commonly distributed at different altitudes (600–3700m above sea level) on Mount Kinabalu. Photosynthetic nitrogen-use efficiency was lower in species at higher altitudes, with a mean PNUE at 3700m being one-third as large as that at 600m. This difference in PNUE was larger than that explained by the biochemical response to lower air pressures only. Across altitudes a negative correlation between ¹³C abundance (¹³C) and PNUE was found. Species at higher altitudes tended to have higher ¹³C, suggesting that they had a lower conductance for CO₂ diffusion from the air to chloroplasts. The lower conductance might be responsible for the lower PNUE in species at higher altitudes. Although leaf nitrogen content per unit area tended to be higher at higher altitudes, it did not seem to contribute to increasing photosynthetic rates. Thus, the idea that a higher nitrogen content at higher altitudes is a compensation for a lower PNUE was not supported. In contrast to the large difference in PNUE among altitudes, PNUE tended to converge within a narrow range among species growing at the same altitude.     

High-level expression and characterization of fully active recombinant conger eel galectins in Eschericia coli

An expression system for recombinant conger eel galectins, congerins I and II, were constructed using the pTV 118N plasmid vector and Escherichia coli. Recombinant congerins I and II could be obtained in the soluble active form with high quantitative yield. Mutation of codons for Val and Leu located in the N-terminal region of Con I increased the expression efficiency. Purification of recombinant proteins were done by only two chromatographical steps from E. coli extract. The purified recombinant congerins were found to be almost the same as the native ones except for the acetyl group at the N-terminus; that is, they showed the same structures and carbohydrate binding activities, suggesting that N-terminal acetyl groups of congerins were not significant for activity.     

Liquid chromatographic-mass spectrometric determination of haloperidol and its metabolites in human plasma and urine

Haloperidol and its two metabolites, reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP) in human plasma and urine were analyzed by HPLC-MS using a new polymer column (MSpak GF-310), which enabled direct injection of crude biological samples without pretreatment. Recoveries of haloperidol and reduced haloperidol spiked into plasma were 64.4-76.1% and 46.8-50.2%, respectively; those for urine were 87.3-99.4% and 94.2-98.5%, respectively; those of CPHP for both samples were not less than 92.7%. The regression equations for haloperidol, reduced haloperidol and CPHP showed good linearity in the ranges of 10-800, 15-800 and 400-800 ng/ml, respectively, for both plasma and urine. Their detection limits were 5, 10 and 300 ng/ml, respectively, for both samples. Thus, the present method was sensitive enough for detection and determination of high therapeutic and toxic levels for haloperidol and its metabolites present in biological samples.     

Single minimum-<Emphasis Type="Italic">B</Emphasis> ambipolar trap

A new type of ambipolar trap is proposed, where a minimum-B magnetic field is used to confine the particles radially and to provide plasma macroscopic stability. The particles are confined axially by creating the plug potentials at both end-mirror cells of the ambipolar trap. The plug potential is produced by only electron cyclotron resonance heating, the mechanism of which is proposed.     

Microbial community changes during organic solid waste treatment analyzed by double gradient-denaturing gradient gel electrophoresis and fluorescence in situ hybridization

The bacterial community present during semicontinuous treatment of organic solid waste under alkaline and high-temperature conditions was studied. PCR-amplified 16S rDNA fragments were analyzed by double gradient-denaturing gradient gel electrophoresis (DGGE). The band pattern was stable during the steady state of the treatment phase, and the major bands resulting from individual treatments had the same DNA sequence with good reproducibility. No sequence in the DNA database of isolated bacteria showed close similarity to this sequence, the closest relative being Bacillus licheniformis with less than 97% similarity. The conditions for fluorescence in situ hybridization (FISH) were determined without the need to obtain extracts of the bacterial cells. An oligonucleotide probe was designed to detect the microorganisms found in the DGGE analysis. FISH analysis showed that the bacterium corresponding to the major bands accounted for 30% of the total eubacterial cell count at the steady state. These results indicate that this bacterium is a key microorganism in the biodegradation process.     

Purification and characterization of the monooxygenase catalyzing sulfur-atom specific oxidation of dibenzothiophene and benzothiophene from the thermophilic bacterium Paenibacillus sp. strain A11-2

A benzothiophene (BT) and dibenzothiophene (DBT) monooxygenase (TdsC), which catalyzes the oxidation of the sulfur atoms in BT and DBT molecules, was purified from Paenibacillus sp. strain A11-2. The molecular mass of the purified enzyme and its subunit were determined to be 200 kDa and 43 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, indicating a tetrameric structure. The N-terminal amino acid sequence of the purified TdsC completely matched the amino acid sequence deduced from the nucleotide sequence of the tdsC gene reported previously [Ishii et al. (2000) Biophys Biochem Res Commun 270:81-88]. The optimal temperature and pH for the TdsC reaction were 65 degrees C and pH 9, respectively. TdsC required NADH, FMN and TdsD, a NADH-dependent FMN oxidoreductase, for its activity, as was observed for TdsA. FAD, lumiflavin and/or NADPH had some effect on the maintenance of TdsC activity. A comparison of the substrate specificity of TdsC and DszC, the homologous monooxygenase purified from Rhodococcus erythropolis strain KA2-5-1, demonstrated a contrasting pattern towards alkylated DBTs and BTs.     

Ethanol modulates gut ischemia/reperfusion-induced liver injury in rats

Whereas both ethanol and gut ischemia/reperfusion (I/R) are known to alter hepatic microvascular function, little is known about the influence of ethanol consumption on the hepatic microvascular responses to I/R. The objective of this study was to determine whether acute ethanol administration exacerbates the hepatic microvascular dysfunction induced by gut I/R. Rats were exposed to gut ischemia for 30 min followed by reperfusion. Intravital videomicroscopy was used to monitor leukocyte recruitment and the number of nonperfused sinusoids (NPS). Plasma alanine aminotransferase (ALT), tumor necrosis factor-alpha (TNF-alpha), and endotoxin concentrations were monitored. In separate experiments, ethanol was administered 15 min or 24 h before gut ischemia. In control rats, gut I/R increased the number of stationary leukocytes and NPS. It also elevated the plasma ALT, TNF-alpha, and endotoxin with a corresponding increase in intestinal mucosal permeability. Low-dose ethanol consumption 15 min before gut ischemia blunted the gut I/R-induced leukostasis and elevations in plasma TNF-alpha and ALT. However, high-dose ethanol consumption aggravated the gut I/R-induced increases in leukostasis and increases in plasma endotoxin and ALT. When ethanol was administered 24 h before, high-dose ethanol aggravated the gut I/R-induced hepatocellular injury, but low-dose ethanol did not have any effects on it. These results suggest that low-dose ethanol consumption shortly before gut ischemia attenuates the hepatic inflammatory responses, microvascular dysfunction, and hepatocellular injury elicited by gut I/R, whereas high-dose ethanol consumption appears to significantly aggravate these gut I/R-induced responses.