Influences of cyclooxygenase inhibitors on the cataleptic behavior induced by haloperidol in mice.

Haloperidol administered intraperitoneally, and prostaglandin F2 alpha (PGF2 alpha) and PGE2 intraventricularly induced dose-dependent cataleptic behavior in mice. The cataleptic behavior induced by haloperidol was inhibited dose-dependently by oral pretreatment with aspirin and indomethacin, inhibitors of PGs synthetase. Striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole 3 acetic acid (5-HIAA) were elevated by haloperidol, although dopamine (DA) and 5-hydroxytryptamine (5-HT) levels did not change. The increase of DOPAC level in striatum induced by haloperidol was significantly suppressed by aspirin, but not in brain stem. The alteration of DOPAC level by aspirin correlated with the behavioral response. These results suggest that central prostaglandin synthesis may participate in the development of cataleptic behavior, which might also involve alteration of brain catecholaminergic activity.     

Reverse hydrolysis reaction of chitin deacetylase and enzymatic synthesis of beta-D-GlcNAc-(1-->4)-GlcN from chitobiose

We found that a chitin deacetylase from Colletotrichum lindemuthianum could acetylate free amino sugar residues into N-acetylated forms in the presence of 3.0 M sodium acetate. The result was analyzed using a beta-N-acetyl-hexosaminidase-coupled assay system with p-nitrophenyl 2-amino-2-deoxy-beta-D-glucopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta- D-glucopyranoside as the substrate, and the liberation of p-nitrophenol was observed as a consequence of enzymatic N-acetylation of the glucosamine residue at the nonreducing end of the substrate. The chitin deacetylase also acetylated chitobiose and chitotetraose as substrates, which was evidenced by the decrease in the amount of free amino sugar residues in the chitooligosaccharides. The reaction product of chitobiose after the acetylation reaction was exclusively 2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->4)-2-amino-2-deoxy-D-gluc ose [GlcNAcGlcN], the structure of which was determined by FABMS and NMR analyses. This study offers a novel method for enzymatic N-acetylation of amino sugars, and especially with chitobiose as substrate, a selectively N-acetylated product, GlcNAcGlcN, can be synthesized.     

Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium,Halomonas elongata

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had K_ms of 9.1 mM for l-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a K_m of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.Halotolerance is of considerable interest scientifically and from the perspective of wide application in fermentation industries and in agriculture. When eubacteria are exposed to hyperosmotic stress, they accumulate various low-molecular-weight organic compounds, the so-called “compatible solutes” such as polyols, amino acids, sugars, and betaines (7–9, 13, 19, 48), because maintenance of turgor pressure is a prerequisite for growth under the conditions of elevated external osmotic pressure. Since Galinski et al. (14) discovered 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) as a compatible solute in Ectothiorhodospira halochloris, an extremely halophilic phototrophic eubacterium, ectoine has been found to be distributed widely in nature, largely in moderately halophilic eubacteria (3, 11, 12, 26, 38, 50). In addition, ectoine has been investigated as a new excellent universal osmoprotectant in this decade, since incorporation of external ectoine under hyperosmotic stress has been observed to confer protection on various nonhalotolerant eubacteria (16, 21, 44).We previously isolated a moderately halophilic eubacterium, Halomonas elongata (31), from dry salty land in Thailand. We identified ectoine and γ-N-acetyl-α,γ-diaminobutyric acid (ADABA), which is one of the cleavage structures of ectoine, as osmotically responding compounds in the cells grown in a glucose-mineral medium containing NaCl in a concentration range of 3 to 15% (31). To understand the accumulation mechanism of the intracellular ectoine, characterization of enzymes involved in the biosynthesis of ectoine is indispensable. Therefore, we have focused on the biosynthetic enzyme of ectoine in this organism. We observed that radioactivity from [1-¹⁴C]aspartate was most efficiently incorporated into ectoine and that the signal intensity was enriched preferentially from [1-¹³C]acetate into the methyl carbon at position 2′ and from [2-¹³C]acetate into the methine carbon at position 2 of the ectoine skeleton, respectively, in ¹³C nuclear magnetic resonance (NMR) spectroscopy (22). From these findings, we also hypothesized the following pathway essentially similar to that described by Peters et al. (34): aspartic β-semialdehyde (ASA) is converted to 2,4-diaminobutyric acid (DABA) by transamination, and DABA is converted to ADABA by acetylation with acetyl coenzyme A (CoA), which in turn yields ectoine by circularization (Fig. ​(Fig.1).1). The three enzymes involved in this pathway are DABA aminotransferase, DABA acetyltransferase, and ectoine synthase in order of the reactions to ectoine. Peters et al. (34) detected the activity of the first and the second of the three steps by using crude extracts of E. halochloris and H. elongata. However, the characterization of these enzymes was limited; in particular, their responses to various salt concentrations remained unknown. Open in a separate windowFIG. 1Proposed biosynthetic pathway of ectoine in H. elongata {"type":"entrez-protein","attrs":{"text":"OUT30018","term_id":"1200192464","term_text":"OUT30018"}}OUT30018.In this study, we confirmed the biosynthetic pathway of ectoine by using purified enzymes in H. elongata {"type":"entrez-protein","attrs":{"text":"OUT30018","term_id":"1200192464","term_text":"OUT30018"}}OUT30018 and characterized the three enzymes involved in the conversion of ASA to ectoine for the first time.     

Treatment of large complex cranial bone defects by using hydroxyapatite ceramic implants

Hydroxyapatite ceramic implants were used in the reconstruction of very large and complex-form cranial bone defects in nine patients. The bone defects were the result of craniectomy after infections and other complications such as severe brain edema, after neurosurgery, and as a result of trauma, subdural hemorrhage, and surgery for brain tumor. The size, shape, and curvature of the hydroxyapatite ceramic implants were determined based on high-precision, full-scale models fabricated through a laser lithographic molding method by using computed tomographic data. The use of this method allowed the fabrication of hydroxyapatite ceramic implants of shapes that accurately matched the area of bone defect, allowing for a minimum of adjustment during the operation even with a complex-form implantation. Not only were good cranial contour reconstructed and aesthetically satisfactory results obtained in the cases treated by incorporating this series of techniques, but neurologic conditions present in some cases were also improved to some extent. The postoperative course has been steady for all nine patients, with no blood transfusions required during or after the operations and no implants requiring removal because of infection or other postoperative complications. The average length of postoperative hospitalization for the nine cases was 11.7 days, remarkably short considering the clinical conditions.     

Crystallographic studies on damaged DNAs. I. An N(6)-methoxyadenine residue forms a Watson-Crick pair with a cytosine residue in a B-DNA duplex

Oxyamines such as hydroxylamine and methoxylamine disturb DNA replication and act as potent mutagens, causing nucleotide transition from one purine to another or one pyrimidine to another. In order to investigate mismatch base-pairing in DNA damaged with oxyamines, a dodecamer with the sequence d(CGCGmo(6)AATCCGCG), where mo(6) A is 2'-deoxy-N(6)-methoxyadenosine, was synthesized and its crystal structure determined. No significant conformational changes are found between the present dodecamer and the original undamaged B-form dodecamer. Electron density maps clearly show that the mo(6)A residue forms a base-pair with a 2'-deoxycytidine residue through hydrogen bonds similar to a Watson-Crick G.C base-pair. For these hydrogen bonds to be made, N(6)-methoxyadenine must chemically take the imino form. The methoxylation thus enables the adenine base to mimic a guanine base. As a result, misincorporation of 2'-deoxycytidine instead of thymidine, or 2'-deoxyadenosine instead of 2'-deoxyguanosine, can occur in DNA replication.     

Mechanism of fluoride action on the L-type calcium channel in cardiac ventricular myocytes

The modulatory actions of fluoride on the function of the dihydropyridine-sensitive (L-type) Ca2+ channel were studied in rabbit cardiac myocytes. In cell-attached voltage-clamp experiments, using barium as the charge carrier, fluoride increased the activity of the Ca2+ channel dose-dependently. Low concentrations (<10 mM) of fluoride increased the number of traces with channel activities, and decreased the number of traces without channel activities, resulting in a net increase in the open-channel probability. The effect of 5 mM fluoride on the Ca2+ channel was inhibited by the presence of non-hydrolyzable guanosine diphosphate analog in the cell. On the other hand, high concentrations (>10 mM) of fluoride increased the open-channel duration, resulting in a marked increase in open-channel probability. A pretreatment of myocytes with a phosphatase inhibitor, okadaic acid, virtually abolished the additional effect of fluoride on the open-channel duration or open probability. A concentration of up to 75 mM fluoride had no effect on the Ca2+-channel activity when the myocytes were pretreated with a potent inhibitor of protein kinases, indicating that fluoride increased the Ca2+- channel activity via modulation of the phosphorylation state of the myocyte or the channel protein alone.     

Effect of climatic conditions on natural mycoflora and fumonisins in freshly harvested corn of the State of Paraná, Brazil

Natural mycoflora associated with fumonisins were analyzed in 150 samples of freshly harvested corn from Central-Southern, Central-Western and Northern regions of the State of Paraná, Brazil and correlated to climatic conditions. The corn samples were frequently contaminated with Fusarium sp.(98.7 to 100%) and Penicillium sp. (93 to 100%), when compared to Aspergillus sp. (not detected to 27.7%). The highest contamination with potentially mycotoxigenic fungi occurred in corn harvested in the Central-Western region, where total mould and yeast counts ranged from 5.5 × 10³ to 5.2 × 10⁶ CFU/g, with 98.7% contaminated byFusarium sp. and 93% by Penicillium sp. In this region F. moniliforme (F. verticillioides) was the predominant Fusariumsp., and was isolated in 85.9% of the samples. Aspergillus sp. was isolated from 27.7% samples. FB₁ was detected in 100% of the samples (mean of 2.39 g/g) and FB₂ in 97.7% (mean of 1.09 g/g). Fumonisins were also detected in all samples from Northern region, with mean of 4.56 g/g (FB₁) and 2.20 g/g (FB₂).Considering 1.0 g/g as the threshold, 72% of the corn samples from the Central-West and 92% from the North were contaminated with concentrations above this value, in contrast to a 18.5% contamination rate from Central-Southern samples. Between corn planting to harvesting season, the average maximum temperature and relative humidity were 26 °C and 77.1%(Central-Southern), 27 °C and 69% (Northern)and 29.9 °C and 89.1% (Central-Western).Therefore, the higher fumonisins contamination of corn from Northern region when compared to the Central-South were due to the differences in rainfall levels (92.8 mm in Central-Southern, 202 mm in Northern) during the month preceding harvest.This revised version was published online in October 2005 with corrections to the Cover Date.     

Improvement in separation of system I and system II particles of photosynthesis obtained by digitonin treatment

By a combined use of digitonin treatment and subsequent centrifugation on a linear sucrose density gradient, the whole green material of the chloroplast lamellae was separated into System I and System II particle fractions, leaving no other fractions of intermediate properties at the final step of separation.

Each of these particle fractions obtained had properties characteristic of System I or System II with respect to the molar ratio of chlorophyll a/chlorophyll b, the content of P700, the fluorescence emission spectrum at −196°;, photoreduction activities with ferricyanide and NADP⁺, and induction of fluorescence.

About 40 and 50% of the total chlorophyll in the original chloroplasts were recovered in System I and System II particles, respectively. Only small amounts of total chlorophyll (less than 10%) were found as free chlorophyll detached from the lamellae through the digitonin treatment.

These results support the view that the lamellae of chloroplasts are composed of about equal amounts of System I and System II particles on a chlorophyll basis.     

Involvement of reactive oxygen species produced via NADPH oxidase in tyrosine phosphorylation in human B- and T-lineage lymphoid cells

In several human B- and T-lymphoid cell lines, reactive oxygen species (ROS) were produced in a time- and dose-dependent manner in response to menadione (vitamin K3) and anti-Fas (CD95/APO-1) mAb when ROS formation was determined by a chemiluminescence-based method. The ROS evoked by menadione and anti-Fas could be first observed as rapidly as within 20 seconds after the stimulation, reaching a maximum within 5-10 min, and declining slowly thereafter. Both menadione and anti-Fas also induced increased tyrosine phosphorylation of multiple cellular proteins whose pattern was similar to that observed upon hydrogen peroxide treatment. For each agent, the kinetics of the increased tyrosine phosphorylation was similar to that of ROS production, and an NADPH oxidase inhibitor, diphenyleneiodonium, prevented both of these two events. Our results suggest a close link between ROS production and tyrosine phosphorylation induced by divergent extracellular stimuli and the possible role of NADPH oxidase or its related enzyme.     

Saccharomyces cerevisiae cultured under aerobic and anaerobic conditions: air-level oxygen stress and protection against stress

Cells of Saccharomyces cerevisiae were grown aerobically and anaerobically, and levels of the protective compounds, cysteine and glutathione, and activities of defensive enzymes, catalase and superoxide dismutase, against an oxygen stress were determined and compared in both cells. Aerobiosis increased both the compounds and enzyme activities. The elevated synthesis of glutathione could be associated with the increased levels of cysteine which in its turn was found to be controlled by the oxygen-dependent activation of cystathionine beta-synthase.