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.     

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.     

High Predation Pressure by an Introduced Flatworm on Land Snails on the Oceanic Ogasawara Islands1

The introduction of a predatory flatworm, Platydemus manokwari, has been considered a cause of the decline of endemic land snails on the tropical oceanic islands. To clarify the effect of P. manokwari on land snail survival in the field, we examined survival rates of snails experimentally placed in areas where snails are absent (i.e., P. manokwari is present) on Chichijima, Ogasawara (Bonin) Islands. We found that over 50 and 90 percent of the snails were dead after 3 and 11 d, respectively, and that the main cause of mortality was predation by P. manokwari.     

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.     

Differential regulation of periplasmic nitrate reductase gene (napKEFDABC) expression between aerobiosis and anaerobiosis with nitrate in a denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans

A denitrifying phototroph, Rhodobacter sphaeroides f. sp. denitrificans, has the ability to denitrify by respiring nitrate. The periplasmic respiratory nitrate reductase (Nap) catalyses the first step in denitrification and is encoded by the genes, napKEFDABC. By assaying the ss-galactosidase activity of napKEFD-lacZ fusions in wild type and nap mutant cells grown under various growth conditions, the environmental signal for inducing nap expression was examined. Under anoxic conditions with nitrate, nap genes expression in the wild-type strain was highest in the dark, and somewhat lowered by incident light, but that of the napA, napB, and napC mutant strains was low, showing that nap expression is dependent on nitrate respiration. Under oxic conditions, both the wild type and nap mutant cells showed high ss-galactosidase activities, comparable to the wild-type grown under anoxic conditions with nitrate. Myxothiazol, a specific inhibitor of the cytochrome bc (1) complex, did not affect the beta-galactosidase activity in the wild-type cells grown aerobically, suggesting that the redox state of the quinone pool was not a candidate for the activation signal for aerobic nap expression. These results suggested that the trans-acting regulatory signals for nap expression differ between anoxic and oxic conditions. Deletion analysis showed that the nucleotide sequence from -135 to -88 with respect to the translational start point is essential for nap expression either under anoxic or oxic conditions, suggesting that the same cis-acting element is involved in regulating nap expression under either anoxic with nitrate or oxic conditions.     

Unusual Accumulation of Demethylspheroidene in Anaerobic-Phototrophic Growth of crtA-Deleted Mutants of Rhodovulum sulfidophilum

Rhodovulum sulfidophilum produces carotenoids in the spheroidene pathway. Spheroidene monooxygenase, CrtA, catalyzes the conversion of spheroidene to spheroidenone. crtA-deleted mutants of R. sulfidophilum did not produce spheroidenone and demethylspheroidenone. In these mutants, the ratio of demethylspheroidene to spheroidene increased with exposure to light. One mutant exhibiting a spheroidene-predominant phenotype did not grow under anaerobic-light conditions and was devoid of bacteriochlorophyll a, even under semiaerobic-light conditions There was no difference in the growth of the mutants under aerobic-dark conditions. These data suggest that demethylspheroidene is important for photosynthesis in R. sulfidophilum.     

The formation of Zn-Chl a in Chlorella heterotrophically grown in the dark with an excessive amount of Zn2+

Chlorella, when heterotrophically cultivated in the dark, is able to grow with Zn2+ at 10-40 mM, which is 10 times the concentration lethal to autotrophically grown cells. However, the lag phase is prolonged with increasing concentrations of Zn2+; for example, in this study, 1 d of the control lag phase was prolonged to about 16 d with Zn2+ at 16.7 mM (x2,000 of the control). Once the cells started to grow, the log phase was finished within 4-6 d regardless of Zn concentration, which was almost the same as that of the control. The photosysystem I reaction center chlorophyll, P700, and the far-red fluorescence were detected only after the late log phase of the growth curve, suggesting that chlorophyll-protein complexes can be organized after cell division has ceased. Interestingly, at more than 16.7 mM of Zn2+, Zn-chlorophyll a was accumulated and finally accounted for about 25% of the total chlorophyll a in the late stationary phase. We found that the Zn-chlorophyll a was present in the thylakoid membranes and not in the soluble fractions of the cells. The rather low fluorescence yield at around 680 nm in the stationary phase suggests that Zn-chlorophyll a can transfer its excitation energy to other chlorophylls. Before accumulation of Zn-chlorophyll a, a marked amount of pheophytin a was temporally accumulated, suggesting that Zn-chlorophyll a could be chemically synthesized via pheophytin a.