Ligand-binding activity and expression profile of annexins in Caenorhabditis elegans

Mammalian annexins are implicated in several physiological mechanisms based on their calcium-dependent phospholipid/membrane binding and carbohydrate-binding activities. In this study, we investigated gene expression profiles of all four Caenorhabditis elegans annexins, nex-1, -2, -3 and -4, throughout the development, and compared phospholipid- and carbohydrate-binding properties of their protein products, NEX-1, -2, -3 and -4. We found that nex-1 and -3 are transcribed continuously during the developmental stages, while expression of nex-2 and -4 appeared to be temporal, peaking at the L1 stage followed by a gradual decrease toward the adult stage. NEX-1 and -3 were detected as single protein band in total worm extracts by immunoblotting, but NEX-2 was heterogenic in size. NEX-1, -2, and -3 showed the binding activities to phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine, but not to phosphatidylcholine. In contrast to their uniform phospholipids-binding properties, their glycosaminoglycan-binding activities were distinctive. NEX-2 bound to heparan sulfate and chondroitin, NEX-3 bound only to heparan sulfate, and NEX-1 showed no lectin activities under tested conditions. NEX-4 had neither phospholipids- nor carbohydrate-binding properties. Differentiated expression profiles and ligand-binding properties of NEX-1, -2, -3 and -4, shown in our study, may represent distinctive roles for each C. elegans annexins.     

Design and synthesis of new potent dipeptidyl peptidase IV inhibitors with enhanced ex vivo duration

A series of 5beta-methylprolyl-2-cyanopyrrolidine analogs were synthesized and evaluated as DPP-IV inhibitors, and the duration of their ex vivo activity was assessed. Comparison of their potency and duration of action was done among three different species. The mode of binding was investigated, and the effect on the plasma glucose level was evaluated. Structure-activity relationships are also presented.     

An expeditious synthesis of tamoxifen, a representative SERM (selective estrogen receptor modulator), via the three-component coupling reaction among aromatic aldehyde, cinnamyltrimethylsilane, and beta-chlorophenetole

Two new synthetic pathways to the anti-cancer agent tamoxifen and its derivatives were developed. The first route involved the aldol reaction of benzyl phenyl ketone with acetaldehyde followed by Friedel–Crafts substitution with anisole in the presence of Cl₂Si(OTf)₂ to produce 1,1,2-triaryl-3-acetoxybutane, a precursor of the tamoxifen derivatives. The second one utilized the novel three-component coupling reaction among aromatic aldehydes, cinnamyltrimethylsilane, and aromatic nucleophiles using HfCl₄ as a Lewis acid catalyst to produce 3,4,4-triarylbutene, that is also a valuable intermediate of the tamoxifen derivatives. The former strategy requires a total of 10 steps from the aldol formation to the final conversion to tamoxifen, whereas the latter needs only three or four steps to produce tamoxifen and droloxifene including the installation of the side-chain moiety and the base-induced double-bond migration to form the tetra-substituted olefin structure. This synthetic strategy seems to serve as a new and practical pathway to prepare not only the tamoxifen derivatives but also the other SERMs (selective estrogen receptor modulators) including estrogen-dependent breast cancer and osteoporosis agents.     

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.     

Laboratory-based evaluation of TOX A/B QUIK CHEK "NISSUI" to detect toxins A and B of clostridium difficile]

The TOX A/B QUIK CHEK "NISSUI" which detects both toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile in stool specimens through immunochromatography was first approved to be released in Japan, and we evaluated its accuracy. In the evaluation, the TOX A/B QUIK CHEK "NISSUI" could correctly detect TcdA and TcdB in solution and in stool specimens spiked with culture broth of TcdA and/or TcdB-producing isolates of C. difficile. The minimum detectable concentrations for TcdA and TcdB were determined to be < or =0.32 ng/ml and < or =0.63 ng/ml, respectively. The TOX A/B QUIK CHEK "NISSUI" gave the consistent results with the colon-endoscopic diagnosis, that is, all the 10 stool specimens from the patients with pseudomembranous colitis were read as being positive, but negative for five patients without any C. difficile-associated disease (CDAD). Of 10 positive stool specimens, one was read as being negative by the commercially available test reagents that can detect only TcdA. In clinical evaluation, a total of 240 stool specimens were tested. Of these, the TOX A/B QUIK CHEK "NISSUI" gave 19 positive results, and TcdA and/or TcdB-producing strains of C. difficile were successfully isolated from all the positive stool specimens, except one. Whereas, of 221 negative stool specimens, 28 isolates of C. difficile were recovered and 11 isolates were identified as TcdA and/or TcdB-producing strains. With these results, it can be concluded that the TOX A/B QUIK CHEK "NISSUI" can correctly detect both TcdA and TcdB of C. difficile, and should be promptly applied to clinical microbiology laboratory to make a definite diagnosis of CDAD, particularly for the CDAD caused by the TcdA-negative but TcdB-positive mutant strains.     

Evaluation of nutritional availability and anti-tumor activity of selenium contained in selenium-enriched Kaiware radish sprouts

We estimated the nutritional availability of selenium (Se) in Se-enriched Kaiware radish sprouts (SeRS) by the tissue Se deposition and glutathione peroxidase (GPX) activity of rats administered the sprouts, and examined the effect of SeRS on the formation of aberrant crypt foci (ACF) in the colon of mice administered 1,2-dimethylhydrazine (DMH) to evaluate anti-tumor activity. Male weanling Wistar rats were divided into seven groups and fed a Se-deficient basal diet or the basal diet supplemented with 0.05, 0.10, or 0.15 microg/g of Se as sodium selenite or SeRS for 28 d. Supplementation with Se dose-dependently increased serum and liver Se concentrations and GPX activities, and the selenite-supplemented groups showed a higher increase than the SeRS-supplemented groups. The nutritional availability of Se in SeRS was estimated to be 33 or 64% by slope ratio analysis. Male 4-week-old A/J mice were divided into seven groups and fed a low Se basal diet or the basal diet supplemented with selenite, SeRS, or selenite + non-Se-enriched radish sprouts (NonSeRS) at a level of 0.1 or 2.0 microg Se/g for 9 weeks. After 1 week of feeding, all mice were given six subcutaneous injections of DMH (20 mg/kg) at 1-week intervals. The average number of ACF formed in the colon of mice fed the basal diet was 4.3. At a supplementation level of 0.1 mug Se/g, only SeRS significantly inhibited ACF formation. At a supplementation level of 2.0 microg Se/g, both selenite and SeRS significantly inhibited ACF formation. The addition of NonSeRS to the selenite-supplemented diets tended to inhibit ACF formation, but this was not statistically significant. These results indicate that SeRS shows lower nutritional availability but higher anti-tumor activity than selenite.     

Monitoring of environmental arsenic by cultures of the photosynthetic bacterial sensor illuminated with a near-infrared light emitting diode array

Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5 microgram/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.     

Role of various carrageenans in autologous and allogeneic mixed lymphocyte reaction

Carrageenans (CGNs) are extracted from cell walls of certain algae of the Rhodophyta and are gel-forming polysaccharides. There are three kinds of purified carrageenans available—kappa, lambda, and iota CGN. All of them stimulated responding T lymphocytes in both autologous and allogeneic mixed lymphocyte reaction, lambda CGN being the most effective. Silica particles abrogated proliferation of responding cells in autologous and allogeneic mixed lymphocyte reaction by killing human monocytes. However, when 1 mg/ml of CGN was added to silica-added assay system, CGN could induce proliferation of responding T cells even in the absence of monocytes. It was concluded that CGN was a monocyte-independent T-lymphocyte mitogen that was also toxic for human monocytes.     

Tetradecane 1,14-Dicarboxylic Acid Production from n-Hexadecane by Candida cloacae

The better condition of cultivation for tetradecane 1,14-dicarboxylic acid (DC-16) production from n-hexadecane (n-C₁₆) by Candida cloacae MR-12 was investigated by using acetic acid as carbon source for the growth. In general, the condition suitable for the growth was also favorable for the production of DC-16. The change of pH during cultivation, the use of NaOH solution as pH controlling agent after pH-change and the addition of antifoam stimulated the production of DC-16.

Under the optimum conditions where the culture medium contained 15% (v/v) n-C₁₆, 1.4% (w/v) acetic acid, inorganic salts and growth factors, and pH was changed from 6.5 to 7.75 at 16 hr after the inoculation, the highest level of DC-16 production was attained after about 72 hr cultivation and the amount of the product accumulated was 61.5 g per liter of the medium.

When a mixture of various n-alkanes was used as starting material, DCs corresponding to the respective n-alkanes were produced as mixture.     

Purification and characterization of β-(pyrazol-1-yl)-l-alanine synthase from Citrullus vulgaris

From seedlings of Citrullus vulgaris the enzyme β-(pyrazol-1-yl)-l-alanine synthase was purified 200-fold, when it showed electrophoretic homogeneity (MW 58 000) and could be dissociated into identical subunits (MW 32 000) each containing one molecule of pyridoxal 5′-phosphate. The K_m value was 2.5 × 10^?3 M for O-acetyl-l-serine and 7.4 × 10^?2 M for pyrazole. The enzyme did not catalyse the formation of related β-substituted alanines, such as l-mimosine and l-quisqualic acid, and significant differences were found between the β-(pyrazol-1-yl)-l-alanine synthase and β-substituted alanine syntheses and cysteine synthase from other sources.