A 1232 bp upstream sequence of glutamine synthetase 1b from Eichhornia crassipes is a root-preferential promoter sequence.

BACKGROUND: Glutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism. It is important to understand the regulation of GS expression in plant. Promoters can initiate the transcription of its downstream gene. Eichhornia crassipes is a most prominent aquatic invasive plant, which has negative effects on environment and economic development. It also can be used in the bioremediation of pollutants present in water and the production of feeding and energy fuel. So identification and characterization of GS promoter in E. crassipes can help to elucidate its regulation mechanism of GS expression and further to control its N metabolism. RESULTS: A 1232 bp genomic fragment upstream of EcGS1b sequence from E. crassipes (EcGS1b-P) has been cloned, analyzed and functionally characterized. TSSP-TCM software and PlantCARE analysis showed a TATA-box core element, a CAAT-box, root specific expression element, light regulation elements including chs-CMA1a, Box I, and Sp1 and other cis-acting elements in the sequence. Three 5'-deletion fragments of EcGS1b upstream sequence with 400 bp, 600 bp and 900 bp length and the 1232 bp fragment were used to drive the expression of ß-glucuronidase (GUS) in tobacco. The quantitative test revealed that GUS activity decreased with the decreasing of the promoter length, which indicated that there were no negative regulated elements in the EcGS1-P. The GUS expressions of EcGS1b-P in roots were significantly higher than those in leaves and stems, indicating EcGS1b-P to be a root-preferential promoter. Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis of EcGS1b gene also showed higher expression in the roots of E.crassipes than in stems and leaves. CONCLUSIONS: EcGS1b-P is a root-preferential promoter sequence. It can specifically drive the transcription of its downstream gene in root. This study will help to elucidate the regulatory mechanisms of EcGS1b tissue-specific expression and further study its other regulatory mechanisms in order to utilize E.crassipes in remediation of eutrophic water and control its overgrowth from the point of nutrient metabolism.

A novel live cell assay to measure diacylglycerol lipase α activity.

Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.

Development of an enzyme assay for rapid assessment of Escherichia coli in seawaters.

AIMS: An analytical protocol has been developed and applied for the detection of glucuronidase activity in marine waters as a rapid alternative approach to assess the microbiological quality of seawaters. METHODS AND RESULTS: The fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide is cleaved to a fluorescent product, methylumbelliferone, by the enzyme beta-glucuronidase, specific to Escherichia coli and closely related enterobacterial species (Shigella). The results suggest that this test is related to E. coli numbers, as estimated by immunofluorescence, more significantly than to faecal coliform numbers, obtained from culture media. CONCLUSIONS: The determination of the potential rate of glucuronidase activity may be used as a diagnostic tool for the indirect estimation of the presence of E. coli in seawaters. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be particularly useful in the early warning of seawater pollution, allowing the screening of coastal areas with different contamination levels in reduced time.

An alternative approach for enumeration of Escherichia coli in foods.

An assay to screen Escherichia coli in foods using MUG supplemented lauryl sulfate tryptose (LST) broth instead of tryptone water (TW) medium was evaluated. The results presented in this paper suggest that this method is more sensitive for lower levels of E. coli, faster (16-18 h vs. 6-10 days) and less expensive (2.454 vs. 2.887 EURO/sample) than the standard ISO procedure. Thus, this method may be beneficial for use when both fecal coliforms and E. coli analyses are required in food systems.

In situ assessment of beta-hexosaminidase activity.

We have adapted two methods to evaluate the beta-hexosaminidase (HEX) enzymatic activity in cultured cells, based on the use of (i) the fluorogenic substrate 4-methylumbelliferyl-6-sulfo-2-acetamido-2- deoxy-beta-D-glucopyranoside (MU-GlcNAc-6-SO4) and (ii) the naphthol AS-BI-N-acetyl-beta-D-glucosaminide and hexazotized pararosaniline. We demonstrate that both methods could be used for the HEX isoenzymes by comparing wild-type and mutant human fibroblast cell lines, deficient for either an alpha or beta subunit from Tay-Sachs and Sandhoff patients. This in situ cytochemical assessment of HEX activity offers a rapid evaluation to study the expression of this enzyme in a heterogeneous cell population such as in gene transfer experiments.

Assessment of the drug inhibitor specificity of the human liver 4-methylumbelliferone UDP-glucuronosyltransferase activity.

The data suggest that the 4MU-UDPGT activity of human liver microsomes probably contributes to the glucuronidation of a limited number of clinically used drugs. However, confirmation of this ultimately requires studies to be performed with purified isozymes, cDNAs expressed in cell culture, or specific inhibitory antibodies.

Rapid identification of Streptococcus bovis by using combination constitutive enzyme substrate hydrolyses.

Several studies have documented the association of blood and rectal-culture positivity for Streptococcus bovis with gastrointestinal neoplasia, especially colonic carcinoma. Conventional methods using bile-esculin hydrolysis, salt tolerance, and sugar fermentations to differentiate S. bovis from other streptococci are laborious, slow, and relatively expensive. Commercially available systems are costly and require at least 24 to 48 h of incubation. A rapid identification procedure for S. bovis and related bacteria was developed. The method uses a reagent containing two hydrolyzable substrates, p-nitrophenyl-alpha-D-galactopyranoside and 4-methylumbilliferyl-beta-D-glucoside, in the presence of 2.5% sodium deoxycholate. This combination test, performed with a rapid assay for L-pyrrolidonyl-aminopeptidase, could distinguish S. bovis, Streptococcus equinus, Enterococcus spp., Streptococcus pneumoniae, and the viridans group streptococci in culture within 30 min. Twelve species of the genera Streptococcus and Enterococcus were tested. The rapid method correlated well with conventional techniques. The reagents are readily available, inexpensive, and easy to make and can be stored in the refrigerator for at least 6 months.